Human β-adrenergic receptor kinase nucleic acid molecule

ABSTRACT

Various embodiments of the invention provide human kinases and phosphatases (KPP) and polynucleotides which identify and encode KPP. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of KPP.

TECHNICAL FIELD

The invention relates to novel nucleic acids, kinases and phosphatasesencoded by these nucleic acids, and to the use of these nucleic acidsand proteins in the diagnosis, treatment, and prevention ofcardiovascular diseases, immune system disorders, neurologicaldisorders, disorders affecting growth and development, lipid disorders,cell proliferative disorders, and cancers. The invention also relates tothe assessment of the effects of exogenous compounds on the expressionof nucleic acids and kinases and phosphatases.

BACKGROUND OF THE INVENTION

Reversible protein phosphorylation is the ubiquitous strategy used tocontrol many of the intracellular events in eukaryotic cells. It isestimated that more than ten percent of proteins active in a typicalmammalian cell are phosphorylated. Kinases catalyze the transfer ofhigh-energy phosphate groups from adenosine triphosphate (ATP) to targetproteins on the hydroxyamino acid residues serine, threonine, ortyrosine. Phosphatases, in contrast, remove these phosphate groups.Extracellular signals including hormones; neurotransmitters, and growthand differentiation factors can activate kinases, which can occur ascell surface receptors or as the activator of the final effectorprotein, as well as other locations along the signal transductionpathway. Cascades of kinases occur, as well as kinases sensitive tosecond messenger molecules. This system allows for the amplification ofweak signals (low abundance growth factor molecules, for example), aswell as the synthesis of many weak signals into an all-or-nothingresponse. Phosphatases, then, are essential in determining the extent ofphosphorylation in the cell and, together with kinases, regulate keycellular processes such as metabolic enzyme activity, proliferation,cell growth and differentiation, cell adhesion, and cell cycleprogression.

Kinases

Kinases comprise the largest known enzyme superfamily and vary widely intheir target molecules. Kinases catalyze the transfer of high energyphosphate groups from a phosphate donor to a phosphate acceptor.Nucleotides usually serve as the phosphate donor in these reactions,with most kinases utilizing adenosine triphosphate (ATP). The phosphateacceptor can be any of a variety of molecules, including nucleosides,nucleotides, lipids, carbohydrates, and proteins. Proteins arephosphorylated on hydroxyamino acids. Addition of a phosphate groupalters the local charge on the acceptor molecule, causing internalconformational changes and potentially influencing intermolecularcontacts. Reversible protein phosphorylation is the primary method forregulating protein activity in eukaryotic cells. In general, proteinsare activated by phosphorylation in response to extracellular signalssuch as hormones, neurotransmitters, and growth and differentiationfactors. The activated proteins initiate the cell's intracellularresponse by way of intracellular signaling pathways and second messengermolecules such as cyclic nucleotides, calcium-calmodulin, inositol, andvarious mitogens, that regulate protein phosphorylation.

Kinases are involved in all aspects of a cell's function, from basicmetabolic processes, such as glycolysis, to cell-cycle regulation,differentiation, and communication with the extracellular environmentthrough signal transduction cascades. Inappropriate phosphorylation ofproteins in cells has been linked to changes in cell cycle progressionand cell differentiation. Changes in the cell cycle have been linked toinduction of apoptosis or cancer. Changes in cell differentiation havebeen linked to diseases and disorders of the reproductive system, immunesystem, and skeletal muscle.

There are two classes of protein kinases. One class, protein tyrosinekinases (PTKs), phosphorylates tyrosine residues, and the other class,protein serine/threonine kinases (STKs), phosphorylates serine andthreonine residues. Some PTKs and STKs possess structuralcharacteristics of both families and have dual specificity for bothtyrosine and serine/threonine residues. Almost all kinases contain aconserved 250-300 amino acid catalytic domain containing specificresidues and sequence motifs characteristic of the kinase family. Theprotein kinase catalytic domain can be further divided into 11subdomains. N-terminal subdomains I-IV fold into a two-lobed structurewhich binds and orients the ATP donor molecule, and subdomain V spansthe two lobes. C-terminal subdomains VI-XI bind the protein substrateand transfer the gamma phosphate from ATP to the hydroxyl group of atyrosine, serine, or threonine residue. Each of the 11 subdomainscontains specific catalytic residues or amino acid motifs characteristicof that subdomain. For example, subdomain I contains an 8-amino acidglycine-rich ATP binding consensus motif, subdomain II contains acritical lysine residue required for maximal catalytic activity, andsubdomains VI through IX comprise the highly conserved catalytic core.PTKs and STKs also contain distinct sequence motifs in subdomains VI andVIII which may confer hydroxyamino acid specificity.

In addition, kinases may also be classified by additional amino acidsequences, generally between 5 and 100 residues, which either flank oroccur within the kinase domain. These additional amino acid sequencesregulate kinase activity and determine substrate specificity. (Reviewedin Hardie, G. and S. Hanks (1995) The Protein Kinase Facts Book, Vol I,pp. 17-20 Academic Press, San Diego Calif.). In particular, two proteinkinase signature sequences have been identified in the kinase domain,the first containing an active site lysine residue involved in ATPbinding, and the second containing an aspartate residue important forcatalytic activity. If a protein analyzed includes the two proteinkinase signatures, the probability of that protein being a proteinkinase is close to 100% (PROSITE: PDOC00100, November 1995).

Protein Tyrosine Kinases

Protein tyrosine kinases (PTKs) may be classified as eithertransmembrane, receptor PTKs or nontransmembrane, nonreceptor PTKproteins. Transmembrane tyrosine kinases function as receptors for mostgrowth factors. Growth factors bind to the receptor tyrosine kinase(RTK), which causes the receptor to phosphorylate itself(autophosphorylation) and specific intracellular second messengerproteins. Growth factors (GF) that associate with receptor PTKs includeepidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, insulinand insulin-like GFs, nerve GF, vascular endothelial GF, and macrophagecolony stimulating factor.

Nontransmembrane, nonreceptor PTKs lack transmembrane regions and,instead, form signaling complexes with the cytosolic domains of plasmamembrane receptors. Receptors that function through non-receptor PTKsinclude those for cytokines and hormones (growth hormone and prolactin),and antigen-specific receptors on T and B lymphocytes.

Many PTKs were first identified as oncogene products in cancer cells inwhich PTK activation was no longer subject to normal cellular controls.In fact, about one third of the known oncogenes encode PTKs.Furthermore, cellular transformation (oncogenesis) is often accompaniedby increased tyrosine phosphorylation activity (Charbonneau, H. and N.K. Tonics (1992) Annu. Rev. Cell Biol. 8:463-493). Regulation of PTKactivity may therefore be an important strategy in controlling sometypes of cancer.

Protein Serine/Threonine Kinases

Protein serine/threonine kinases (STKs) are nontransmembrane proteins. Asubclass of STKs are known as ERKs (extracellular signal regulatedkinases) or MAPs (mitogen-activated protein kinases) and are activatedafter cell stimulation by a variety of hormones and growth factors. Cellstimulation induces a signaling cascade leading to phosphorylation ofMEK (MAP/ERK kinase) which, in turn, activates ERK via serine andthreonine phosphorylation. A varied number of proteins represent thedownstream effectors for the active ERK and implicate it in the controlof cell proliferation and differentiation, as well as regulation of thecytoskeleton. Activation of ERK is normally transient, and cells possessdual specificity phosphatases that are responsible for itsdown-regulation. Also, numerous studies have shown that elevated ERKactivity is associated with some cancers. Other STKs include the secondmessenger dependent protein kinases such as the cyclic-AMP dependentprotein kinases (PKA), calcium-calmodulin (CaM) dependent proteinkinases, and the mitogen-activated protein kinases (MAP); thecyclin-dependent protein kinases; checkpoint and cell cycle kinases;Numb-associated kinase (Nak); human Fused (hFu); proliferation-relatedkinases; 5′-AMP-activated protein kinases; and kinases involved inapoptosis.

One member of the ERK family of MAP kinases, ERK 7, is a novel 61-kDaprotein that has motif similarities to ERK1 and ERK2, but is notactivated by extracellular stimuli as are ERK1 and ERK2 nor by thecommon activators, c-Jun N-terminal kinase (JNK) and p38 kinase. ERK7regulates its nuclear localization and inhibition of growth through itsC-terminal tail, not through the kinase domain as is typical with otherMAP kinases (Abe, M. K. (1999) Mol. Cell. Biol. 19:1301-1312).

The second messenger dependent protein kinases primarily mediate theeffects of second messengers such as cyclic AMP (cAMP), cyclic GMP,inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclicADP ribose, arachidonic acid, diacylglycerol and calcium-calmodulin. ThePKAs are involved in mediating hormone-induced cellular responses andare activated by cAMP produced within the cell in response to hormonestimulation. cAMP is an intracellular mediator of hormone action in allanimal cells that have been studied. Hormone-induced cellular responsesinclude thyroid hormone secretion, cortisol secretion, progesteronesecretion, glycogen breakdown, bone resorption, and regulation of heartrate and force of heart muscle contraction. PKA is found in all animalcells and is thought to account for the effects of cAMP in most of thesecells. Altered PKA expression is implicated in a variety of disordersand diseases including cancer, thyroid disorders, diabetes,atherosclerosis, and cardiovascular disease (Isselbacher, K. J. et al.(1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New YorkN.Y., pp. 416-431, 1887).

The casein kinase I (CKI) gene family is another subfamily ofserine/threonine protein kinases. This continuously expanding group ofkinases have been implicated in the regulation of numerous cytoplasmicand nuclear processes, including cell metabolism and DNA replication andrepair. CKI enzymes are present in the membranes, nucleus, cytoplasm andcytoskeleton of eukaryotic cells, and on the mitotic spindles ofmammalian cells (Fish, K. J. et al. (1995) J. Biol. Chem.270:14875-14883).

The CKI family members all have a short amino-terminal domain of 9-76amino acids, a highly conserved kinase domain of 284 amino acids, and avariable carboxyl-terminal domain that ranges from 24 to over 200 aminoacids in length (Cegielska, A. et al. (1998) J. Biol. Chem.273:1357-1364). The CKI family is comprised of highly related proteins,as seen by the identification of isoforms of casein kinase I from avariety of sources. There are at least five mammalian isoforms, α, β, γ,δ, and ε. Fish et al. identified CKI-epsilon from a human placenta cDNAlibrary. It is a basic protein of 416 amino acids and is closest toCKI-delta. Through recombinant expression, it was determined tophosphorylate known CKI substrates and was inhibited by the CKI-specificinhibitor CKI-7. The human gene for CKI-epsilon was able to rescue yeastwith a slow-growth phenotype caused by deletion of the yeast CKI locus,HRR250 (Fish et al., supra).

The mammalian circadian mutation tau was found to be a semidominantautosomal allele of CKI-epsilon that markedly shortens period length ofcircadian rhythms in Syrian hamsters. The tau locus is encoded by caseinkinase I-epsilon, which is also a homolog of the Drosophila circadiangene double-time. Studies of both the wildtype and tau mutantCKI-epsilon enzyme indicated that the mutant enzyme has a noticeablereduction in the maximum velocity and autophosphorylation state.Further, in vitro, CKI-epsilon is able to interact with mammalian PERIODproteins, while the mutant enzyme is deficient in its ability tophosphorylate PERIOD. Lowrey et al. have proposed that CKI-epsilon playsa major role in delaying the negative feedback signal within thetranscription-translation-based autoregulatory loop that composes thecore of the circadian mechanism. Therefore the CKI-epsilon enzyme is anideal target for pharmaceutical compounds influencing circadian rhythms,jet-lag and sleep, in addition to other physiologic and metabolicprocesses under circadian regulation (Lowrey, P. L. et al. (2000)Science 288:483-491).

Homeodomain-interacting protein kinases (HIPKs) are serine/threoninekinases and novel members of the DYRK kinase subfamily (Hofmann, T. G.et al. (2000) Biochimie 82:1123-1127). HIPKs contain a conserved proteinkinase domain separated from a domain that interacts with homeoproteins.HIPKs are nuclear kinases, and HIPK2 is highly expressed in neuronaltissue (Kim, Y. H. et al. (1998) J. Biol. Chem. 273:25875-25879; Wang,Y. et al. (2001) Biochim. Biophys. Acta 1518:168-172). HIPKs act ascorepressors for homeodomian transcription factors. This corepressoractivity is seen in posttranslational modifications such asubiquitination and phosphorylation, each of which are important in theregulation of cellular protein function (Kim, Y. H. et al. (1999) Proc.Natl. Acad. Sci. USA 96:12350-12355).

The human h-warts protein, a homolog of Drosophila warts tumorsuppressor gene, maps to chromosome 6q24-25.1. It has a serine/threoninekinase domain and is localized to centrosomes in interphase cells. It isinvolved in mitosis and functions as a component of the mitoticapparatus (Nishiyama, Y. et al. (1999) FEBS Lett. 459:159-165).

Calcium-Calmodulin Dependent Protein Kinases

Calcium-calmodulin dependent (CaM) kinases are involved in regulation ofsmooth muscle contraction, glycogen breakdown (phosphorylase kinase),and neurotransmission (CaM kinase I and CaM kinase II). CaM dependentprotein kinases are activated by calmodulin, an intracellular calciumreceptor, in response to the concentration of free calcium in the cell.Many CaM kinases are also activated by phosphorylation. Some CaM kinasesare also activated by autophosphorylation or by other regulatorykinases. CaM kinase I phosphorylates a variety of substrates includingthe neurotransmitter-related proteins synapsin I and II, the genetranscription regulator, CREB, and the cystic fibrosis conductanceregulator protein, CFTR (Haribabu, B. et al. (1995) EMBO J.14:3679-3686). CaM kinase II also phosphorylates synapsin at differentsites and controls the synthesis of catecholamines in the brain throughphosphorylation and activation of tyrosine hydroxylase. CaM kinase IIcontrols the synthesis of catecholamines and seratonin, throughphosphorylation/activation of tyrosine hydroxylase and tryptophanhydroxylase, respectively (Fujisawa, H. (1990) BioEssays 12:27-29). ThemRNA encoding a calmodulin-binding protein kinase-like protein was foundto be enriched in mammalian forebrain. This protein is associated withvesicles in both axons and dendrites and accumulates largelypostnatally. The amino acid sequence of this protein is similar toCaM-dependent STKs, and the protein binds calmodulin in the presence ofcalcium (Godbout, M. et al. (1994) J. Neurosci. 14:1-13).

Mitogen-Activated Protein Kinases

The mitogen-activated protein kinases (MAP), which mediate signaltransduction from the cell surface to the nucleus via phosphorylationcascades, are another STK family that regulates intracellular signalingpathways. Several subgroups have been identified, and each manifestsdifferent substrate specificities and responds to distinct extracellularstimuli (Egan, S. E. and R. A. Weinberg (1993) Nature 365:781-783).There are three kinase modules comprising the MAP kinase cascade: MAPK(MAP), MAPK kinase (MAP2K, MAPKK, or MKK), and MKK kinase (MAP3K,MAPKKK, OR MEKK) (Wang, X. S. et al (1998) Biochem. Biophys. Res.Commun. 253:33-37). The extracellular-regulated kinase (ERK) pathway isactivated by growth factors and mitogens, for example, epidermal growthfactor (EGF), ultraviolet light, hyperosmolar medium, heat shock, orendotoxic lipopolysaccharide (LPS). The closely related though distinctparallel pathways, the c-Jun N-terminal kinase (JNK), orstress-activated kinase (SAPK) pathway, and the p38 kinase pathway areactivated by stress stimuli and proinflammatory cytokines such as tumornecrosis factor (TNF) and interleukin-1 (IL-1). Altered MAP kinaseexpression is implicated in a variety of disease conditions includingcancer, inflammation, immune disorders, and disorders affecting growthand development. MAP kinase signaling pathways are present in mammaliancells as well as in yeast.

Cyclin-Dependent Protein Kinases

The cyclin-dependent protein kinases (CDKs) are STKs that control theprogression of cells through the cell cycle. The entry and exit of acell from mitosis are regulated by the synthesis and destruction of afamily of activating proteins called cyclins. Cyclins are smallregulatory proteins that bind to and activate CDKs, which thenphosphorylate and activate selected proteins involved in the mitoticprocess. CDKs are unique in that they require multiple inputs to becomeactivated. In addition to cyclin binding, CDK activation requires thephosphorylation of a specific threonine residue and thedephosphorylation of a specific tyrosine residue on the CDK.

Another family of STKs associated with the cell cycle are the NIMA(never in mitosis)-related kinases (Neks). Both CDKs and Neks areinvolved in duplication, maturation, and separation of the microtubuleorganizing center, the centrosome, in animal cells (Fry, A. M. et al.(1998) EMBO J. 17:470-481).

Checkpoint and Cell Cycle Kinases

In the process of cell division, the order and timing of cell cycletransitions are under control of cell cycle checkpoints, which ensurethat critical events such as DNA replication and chromosome segregationare carried out with precision. If DNA is damaged, e.g. by radiation, acheckpoint pathway is activated that arrests the cell cycle to providetime for repair. If the damage is extensive, apoptosis is induced. Inthe absence of such checkpoints, the damaged DNA is inherited byaberrant cells which may cause proliferative disorders such as cancer.Protein kinases play an important role in this process. For example, aspecific kinase, checkpoint kinase 1 (Chk1), has been identified inyeast and mammals, and is activated by DNA damage in yeast. Activationof Chk1 leads to the arrest of the cell at the G2/M transition (Sanchez,Y. et al. (1997) Science 277:1497-1501). Specifically, Chk1phosphorylates the cell division cycle phosphatase CDC25, inhibiting itsnormal function which is to dephosphorylate and activate thecyclin-dependent kinase Cdc2. Cdc2 activation controls the entry ofcells into mitosis (Peng, C.-Y. et al. (1997) Science 277:1501-1505).Thus, activation of Chk1 prevents the damaged cell from enteringmitosis. A deficiency in a checkpoint kinase, such as Chk1, may alsocontribute to cancer by failure to arrest cells with damaged DNA atother checkpoints such as G2/M.

Proliferation-Related Kinases

Proliferation-related kinase is a serum/cytokine inducible STK that isinvolved in regulation of the cell cycle and cell proliferation in humanmegakarocytic cells (Li, B. et al. (1996) J. Biol. Chem.271:19402-19408). Proliferation-related kinase is related to the polo(derived from Drosophila polo gene) family of STKs implicated in celldivision. Proliferation-related kinase is downregulated in lung tumortissue and may be a proto-oncogene whose deregulated expression innormal tissue leads to oncogenic transformation.

5′-AMP-Activated Protein Kinase

A ligand-activated STK protein kinase is 5′-AMP-activated protein kinase(AMPK) (Gao, G. et al. (1996) J. Biol. Chem. 271:8675-8681). MammalianAMPK is a regulator of fatty acid and sterol synthesis throughphosphorylation of the enzymes acetyl-CoA carboxylase andhydroxymethylglutaryl-CoA reductase and mediates responses of thesepathways to cellular stresses such as heat shock and depletion ofglucose and ATP. AMPK is a heterotrimeric complex comprised of acatalytic alpha subunit and two non-catalytic beta and gamma subunitsthat are believed to regulate the activity of the alpha subunit.Subunits of AMPK have a much wider distribution in non-lipogenic tissuessuch as brain, heart, spleen, and lung than expected. This distributionsuggests that its role may extend beyond regulation of lipid metabolismalone.

Kinases in Apoptosis

Apoptosis is a highly regulated signaling pathway leading to cell deaththat plays a crucial role in tissue development and homeostasis.Deregulation of this process is associated with the pathogenesis of anumber of diseases including autoimmune diseases, neurodegenerativedisorders, and cancer. Various STKs play key roles in this process. ZIPkinase is an STK containing a C-terminal leucine zipper domain inaddition to its N-terminal protein kinase domain. This C-terminal domainappears to mediate homodimerization and activation of the kinase as wellas interactions with transcription factors such as activatingtranscription factor, ATF4, a member of the cyclic-AMP responsiveelement binding protein (ATF/CREB) family of transcriptional factors(Sanjo, H. et al. (1998) J. Biol. Chem. 273:29066-29071). DRAK1 andDRAK2 are STKs that share homology with the death-associated proteinkinases (DAP kinases), known to function in interferon-γ inducedapoptosis (Sanjo et al., supra). Like ZIP kinase, DAP kinases contain aC-terminal protein-protein interaction domain, in the form of ankyrinrepeats, in addition to the N-terminal kinase domain. ZIP, DAP, and DRAKkinases induce morphological changes associated with apoptosis whentransfected into NIH3T3 cells (Sanjo et al., supra). However, deletionof either the N-terminal kinase catalytic domain or the C-terminaldomain of these proteins abolishes apoptosis activity, indicating thatin addition to the kinase activity, activity in the C-terminal domain isalso necessary for apoptosis, possibly as an interacting domain with aregulator or a specific substrate.

RICK is another STK recently identified as mediating a specificapoptotic pathway involving the death receptor, CD95 (Inohara, N. et al.(1998) J. Biol. Chem. 273:12296-12300). CD95 is a member of the tumornecrosis factor receptor superfamily and plays a critical role in theregulation and homeostasis of the immune system (Nagata, S. (1997) Cell88:355-365). The CD95 receptor signaling pathway involves recruitment ofvarious intracellular molecules to a receptor complex following ligandbinding. This process includes recruitment of the cysteine proteasecaspase-8 which, in turn, activates a caspase cascade leading to celldeath. RICK is composed of an N-terminal kinase catalytic domain and aC-terminal “caspase-recruitment” domain that interacts with caspase-likedomains, indicating that RICK plays a role in the recruitment ofcaspase-8. This interpretation is supported by the fact that theexpression of RICK in human 293T cells promotes activation of caspase-8and potentiates the induction of apoptosis by various proteins involvedin the CD95 apoptosis pathway (Inohara et al., supra).

Mitochondrial Protein Kinases

A novel class of eukaryotic kinases, related by sequence to prokaryotichistidine protein kinases, are the mitochondrial protein kinases (MPKs)which seem to have no sequence similarity with other eukaryotic proteinkinases. These protein kinases are located exclusively in themitochondrial matrix space and may have evolved from genes originallypresent in respiration-dependent bacteria which were endocytosed byprimitive eukaryotic cells. MPKs are responsible for phosphorylation andinactivation of the branched-chain alpha-ketoacid dehydrogenase andpyruvate dehydrogenase complexes (Harris, R. A. et al. (1995) Adv.Enzyme Regul. 34:147-162). Five MPKs have been identified. Four memberscorrespond to pyruvate dehydrogenase kinase isozymes, regulating theactivity of the pyruvate dehydrogenase complex, which is an importantregulatory enzyme at the interface between glycolysis and the citricacid cycle. The fifth member corresponds to a branched-chainalpha-ketoacid dehydrogenase kinase, important in the regulation of thepathway for the disposal of branched-chain amino acids. (Harris, R. A.et al. (1997) Adv. Enzyme Regul. 37:271-293). Both starvation and thediabetic state are known to result in a great increase in the activityof the pyruvate dehydrogenase kinase in the liver, heart and muscle ofthe rat. This increase contributes in both disease states to thephosphorylation and inactivation of the pyruvate dehydrogenase complexand conservation of pyruvate and lactate for gluconeogenesis (Harris(1995) supra).

Kinases with Non-Protein Substrates

Lipid and Inositol Kinases

Lipid kinases phosphorylate hydroxyl residues on lipid head groups. Afamily of kinases involved in phosphorylation of phosphatidylinositol(PI) has been described, each member phosphorylating a specific carbonon the inositol ring (Leevers, S. J. et al. (1999) Curr. Opin. Cell.Biol. 11:219-225). The phosphorylation of phosphatidylinositol isinvolved in activation of the protein kinase C signaling pathway. Theinositol phospholipids (phosphoinositides) intracellular signalingpathway begins with binding of a signaling molecule to a G-proteinlinked receptor in the plasma membrane. This leads to thephosphorylation of phosphatidylinositol (PI) residues on the inner sideof the plasma membrane by inositol kinases, thus converting PI residuesto the biphosphate state (PIP₂). PIP₂ is then cleaved into inositoltriphosphate (IP₃) and diacylglycerol. These two products act asmediators for separate signaling pathways. Cellular responses that aremediated by these pathways are glycogen breakdown in the liver inresponse to vasopressin, smooth muscle contraction in response toacetylcholine, and thrombin-induced platelet aggregation.

PI 3-kinase (PI3K), which phosphorylates the D3 position of PI and itsderivatives, has a central role in growth factor signal cascadesinvolved in cell growth, differentiation, and metabolism. PI3K is aheterodimer consisting of an adapter subunit and a catalytic subunit.The adapter subunit acts as a scaffolding protein, interacting withspecific tyrosine-phosphorylated proteins, lipid moieties, and othercytosolic factors. When the adapter subunit binds tyrosinephosphorylated targets, such as the insulin responsive substrate(IRS)-1, the catalytic subunit is activated and converts PI (4,5)bisphosphate (PIP₂) to PI (3,4,5) P₃ (PIP₃). PIP₃ then activates anumber of other proteins, including PKA, protein kinase B (PKB), proteinkinase C (PKC), glycogen synthase kinase (GSK)-3, and p70 ribosomal s6kinase. PI3K also interacts directly with the cytoskeletal organizingproteins, Rac, rho, and cdc42 (Shepherd, P. R. et al (1998) Biochem. J.333:471-490) Animal models for diabetes, such as obese and fat mice,have altered PI3K adapter subunit levels. Specific mutations in theadapter subunit have also been found in an insulin-resistant Danishpopulation, suggesting a role for PI3K in type-2 diabetes (Shepard,supra).

An example of lipid kinase phosphorylation activity is thephosphorylation of D-erythro-sphingosine to the sphingolipid metabolite,sphingosine-1-phosphate (SPP). SPP has emerged as a novel lipidsecond-messenger with both extracellular and intracellular actions(Kohama, T. et al. (1998) J. Biol. Chem. 273:23722-23728).Extracellularly, SPP is a ligand for the G-protein coupled receptorEDG-1 (endothelial-derived, G-protein coupled receptor).Intracellularly, SPP regulates cell growth, survival, motility, andcytoskeletal changes. SPP levels are regulated by sphingosine kinasesthat specifically phosphorylate D-erythro-sphingosine to SPP. Theimportance of sphingosine kinase in cell signaling is indicated by thefact that various stimuli, including platelet-derived growth factor(PDGF), nerve growth factor, and activation of protein kinase C,increase cellular levels of SPP by activation of sphingosine kinase, andthe fact that competitive inhibitors of the enzyme selectively inhibitcell proliferation induced by PDGF (Kohama et al., supra).

Purine Nucleotide Kinases

The purine nucleotide kinases, adenylate kinase (ATP:AMPphosphotransferase, or AdK) and guanylate kinase (ATP:GMPphosphotransferase, or GuK) play a key role in nucleotide metabolism andare crucial to the synthesis and regulation of cellular levels of ATPand GTP, respectively. These two molecules are precursors in DNA and RNAsynthesis in growing cells and provide the primary source of biochemicalenergy in cells (ATP), and signal transduction pathways (GTP).Inhibition of various steps in the synthesis of these two molecules hasbeen the basis of many antiproliferative drugs for cancer and antiviraltherapy (Pillwein, K. et al. (1990) Cancer Res. 50:1576-1579).

AdK is found in almost all cell types and is especially abundant incells having high rates of ATP synthesis and utilization such asskeletal muscle. In these cells AdK is physically associated withmitochondria and myofibrils, the subcellular structures that areinvolved in energy production and utilization, respectively. Recentstudies have demonstrated a major function for AdK in transferring highenergy phosphoryls from metabolic processes generating ATP to cellularcomponents consuming ATP (Zeleznikar, R. J. et al. (1995) J. Biol. Chem.270:7311-7319). Thus AdK may have a pivotal role in maintaining energyproduction in cells, particularly those having a high rate of growth ormetabolism such as cancer cells, and may provide a target forsuppression of its activity in order to treat certain cancers.Alternatively, reduced AdK activity may be a source of variousmetabolic, muscle-energy disorders that can result in cardiac orrespiratory failure and may be treatable by increasing AdK activity.

GuK, in addition to providing a key step in the synthesis of GTP for RNAand DNA synthesis, also fulfills an essential function in signaltransduction pathways of cells through the regulation of GDP and GTP.Specifically, GTP binding to membrane associated G proteins mediates theactivation of cell receptors, subsequent intracellular activation ofadenyl cyclase, and production of the second messenger, cyclic AMP. GDPbinding to G proteins inhibits these processes. GDP and GTP levels alsocontrol the activity of certain oncogenic proteins such as p21′ known tobe involved in control of cell proliferation and oncogenesis (Bos, J. L.(1989) Cancer Res. 49:4682-4689). High ratios of GTP:GDP caused bysuppression of GuK cause activation of p21^(ras) and promoteoncogenesis. Increasing GuK activity to increase levels of GDP andreduce the GTP:GDP ratio may provide a therapeutic strategy to reverseoncogenesis.

GuK is an important enzyme in the phosphorylation and activation ofcertain antiviral drugs useful in the treatment of herpes virusinfections. These drugs include the guanine homologs acyclovir andbuciclovir (Miller, W. H. and R. L. Miller (1980) J. Biol. Chem.255:7204-7207; Stenberg, K. et al. (1986) J. Biol. Chem. 261:2134-2139).Increasing GuK activity in infected cells may provide a therapeuticstrategy for augmenting the effectiveness of these drugs and possiblyfor reducing the necessary dosages of the drugs.

Pyrimidine Kinases

The pyrimidine kinases are deoxycytidine kinase and thymidine kinase 1and 2. Deoxycytidine kinase is located in the nucleus, and thymidinekinase 1 and 2 are found in the cytosol (Johansson, M. et al. (1997)Proc. Natl. Acad. Sci. USA 94:11941-11945). Phosphorylation ofdeoxyribonucleosides by pyrimidine kinases provides an alternativepathway for de novo synthesis of DNA precursors. The role of pyrimidinekinases, like purine kinases, in phosphorylation is critical to theactivation of several chemotherapeutically important nucleosideanalogues (Arner E. S. and S. Eriksson (1995) Pharmacol. Ther.67:155-186).

Phosphatases

Protein phosphatases are generally characterized as eitherserine/threonine- or tyrosine-specific based on their preferredphospho-amino acid substrate. However, some phosphatases (DSPs, for dualspecificity phosphatases) can act on phosphorylated tyrosine, serine, orthreonine residues. The protein serine/threonine phosphatases (PSPs) areimportant regulators of many cAMP-mediated hormone responses in cells.Protein tyrosine phosphatases (PTPs) play a significant role in cellcycle and cell signaling processes. Another family of phosphatases isthe acid phosphatase or histidine acid phosphatase (HAP) family whosemembers hydrolyze phosphate esters at acidic pH conditions.

PSPs are found in the cytosol, nucleus, and mitochondria and inassociation with cytoskeletal and membranous structures in most tissues,especially the brain. Some PSPs require divalent cations, such as Ca²⁺or Mn²⁺, for activity. PSPs play important roles in glycogen metabolism,muscle contraction, protein synthesis, T cell function, neuronalactivity, oocyte maturation, and hepatic metabolism (reviewed in Cohen,P. (1989) Annu. Rev. Biochem. 58:453-508). PSPs can be separated intotwo classes. The PPP class includes PP1, PP2A, PP2B/calcineurin, PP4,PP5, PP6, and PP7. Members of this class are composed of a homologouscatalytic subunit bearing a very highly conserved signature sequence,coupled with one or more regulatory subunits (PROSITE PDOC00115).Further interactions with scaffold and anchoring molecules determine theintracellular localization of PSPs and substrate specificity. The PPMclass consists of several closely related isoforms of PP2C and isevolutionarily unrelated to the PPP class.

PP1 dephosphorylates many of the proteins phosphorylated by cyclicAMP-dependent protein kinase (PKA) and is an important regulator of manycAMP-mediated hormone responses in cells. A number of isoforms have beenidentified, with the alpha and beta forms being produced by alternativesplicing of the same gene. Both ubiquitous and tissue-specific targetingproteins for PP1 have been identified. In the brain, inhibition of PP1activity by the dopamine and adenosine 3′,5′-monophosphate-regulatedphosphoprotein of 32kDa (DARPP-32) is necessary for normal dopamineresponse in neostriatal neurons (reviewed in Price, N. E. and M. C.Mumby (1999) Curr. Opin. Neurobiol. 9:336-342). PP1, along with PP2A,has been shown to limit motility in microvascular endothelial cells,suggesting a role for PSPs in the inhibition of angiogenesis (Gabel, S.et al. (1999) Otolaryngol. Head Neck Surg. 121:463-468).

PP2A is the main serine/threonine phosphatase. The core PP2A enzymeconsists of a single 36 kDa catalytic subunit (C) associated with a 65kDa scaffold subunit (A), whose role is to recruit additional regulatorysubunits (B). Three gene families encoding B subunits are known (PR55,PR61, and PR72), each of which contain multiple isoforms, and additionalfamilies may exist (Millward, T. A et al. (1999) Trends Biosci.24:186-191). These “B-type” subunits are cell type- and tissue-specificand determine the substrate specificity, enzymatic activity, andsubcellular localization of the holoenzyme. The PR55 family is highlyconserved and bears a conserved motif (PROSITE PDOC00785). PR55increases PP2A activity toward mitogen-activated protein kinase (MAPK)and MAPK kinase (MEK). PP2A dephosphorylates the MAPK active site,inhibiting the cell's entry into mitosis. Several proteins can competewith PR55 for PP2A core enzyme binding, including the CKII kinasecatalytic subunit, polyomavirus middle and small T antigens, and SV40small t antigen. Viruses may use this mechanism to commandeer PP2A andstimulate progression of the cell through the cell cycle (Pallas, D. C.et al. (1992) J. Virol. 66:886-893). Altered MAP kinase expression isalso implicated in a variety of disease conditions including cancer,inflammation, immune disorders, and disorders affecting growth anddevelopment. PP2A, in fact, can dephosphorylate and modulate theactivities of more than 30 protein kinases in vitro, and other evidencesuggests that the same is true in vivo for such kinases as PKB, PKC, thecalmodulin-dependent kinases, ERK family MAP kinases, cyclin-dependentkinases, and the IκB kinases (reviewed in Millward et al., supra). PP2Ais itself a substrate for CKI and CKII kinases, and can be stimulated bypolycationic macromolecules. A PP2A-like phosphatase is necessary tomaintain the G1 phase destruction of mammalian cyclins A and B(Bastians, H. et al. (1999) Mol. Biol. Cell 10:3927-3941). PP2A is amajor activity in the brain and is implicated in regulatingneurofilament stability and normal neural function, particularly thephosphorylation of the microtubule-associated protein tau.Hyperphosphorylation of tau has been proposed to lead to the neuronaldegeneration seen in Alzheimer's disease (reviewed in Price and Mumby,supra).

PP2B, or calcineurin, is a Ca²⁺-activated dimeric phosphatase and isparticularly abundant in the brain. It consists of catalytic andregulatory subunits, and is activated by the binding of thecalcium/calmodulin complex. Calcineurin is the target of theimmunosuppressant drugs cyclosporine and FK506. Along with othercellular factors, these drugs interact with calcineurin and inhibitphosphatase activity. In T cells, this blocks the calcium dependentactivation of the NF-AT family of transcription factors, leading toimmunosuppression. This family is widely distributed, and it is likelythat calcineurin regulates gene expression in other tissues as well. Inneurons, calcineurin modulates functions which range from the inhibitionof neurotransmitter release to desensitization of postsynapticNMDA-receptor coupled calcium channels to long term memory (reviewed inPrice and Mumby, supra).

Other members of the PPP class have recently been identified (Cohen, P.T. (1997) Trends Biochem. Sci. 22:245-251). One of them, PP5, containsregulatory domains with tetratricopeptide repeats. It can be activatedby polyunsaturated fatty acids and anionic phospholipids in vitro andappears to be involved in a number of signaling pathways, includingthose controlled by atrial natriuretic peptide or steroid hormones(reviewed in Andreeva, A. V. and M. A. Kutuzov (1999) Cell Signal.11:555-562).

PP2C is a ˜42 kDa monomer with broad substrate specificity and isdependent on divalent cations (mainly Me or Me) for its activity. PP2Cproteins share a conserved N-terminal region with an invariant DGHmotif, which contains an aspartate residue involved in cation binding(PROSITE PDOC00792). Targeting proteins and mechanisms regulating PP2Cactivity have not been identified. PP2C has been shown to inhibit thestress-responsive p38 and Jun kinase (JNK) pathways (Takekawa, M. et al.(1998) EMBO J. 17:4744-4752).

In contrast to PSPs, tyrosine-specific phosphatases (PTPs) are generallymonomeric proteins of very diverse size (from 20 kDa to greater than 100kDa) and structure that function primarily in the transduction ofsignals across the plasma membrane. PTPs are categorized as eithersoluble phosphatases or transmembrane receptor proteins that contain aphosphatase domain. All PTPs share a conserved catalytic domain of about300 amino acids which contains the active site. The active siteconsensus sequence includes a cysteine residue which executes anucleophilic attack on the phosphate moiety during catalysis (Neel, B.G. and N. K. Tonics (1997) Curr. Opin. Cell Biol. 9:193-204). ReceptorPTPs are made up of an N-terminal extracellular domain of variablelength, a transmembrane region, and a cytoplasmic region that generallycontains two copies of the catalytic domain. Although only the firstcopy seems to have enzymatic activity, the second copy apparentlyaffects the substrate specificity of the first. The extracellulardomains of some receptor PTPs contain fibronectin-like repeats,immunoglobulin-like domains, MAM domains (an extracellular motif likelyto have an adhesive function), or carbonic anhydrase-like domains(PROSITE PDOC 00323). This wide variety of structural motifs accountsfor the diversity in size and specificity of PTPs.

PTPs play important roles in biological processes such as cell adhesion,lymphocyte activation, and cell proliferation. PTPs μ and κ are involvedin cell-cell contacts, perhaps regulating cadherin/catenin function. Anumber of PTPs affect cell spreading, focal adhesions, and cellmotility, most of them via the integrin/tyrosine kinase signalingpathway (reviewed in Neel and Tonics, supra). CD45 phosphatases regulatesignal transduction and lymphocyte activation (Ledbetter, J. A. et al.(1988) Proc. Natl. Acad. Sci. USA 85:8628-8632). Soluble PTPs containingSrc-homology-2 domains have been identified (SHPs), suggesting thatthese molecules might interact with receptor tyrosine kinases. SHP-1regulates cytokine receptor signaling by controlling the Janus familyPTKs in hematopoietic cells, as well as signaling by the T-cell receptorand c-Kit (reviewed in Neel and Tonics, supra). M-phase inducerphosphatase plays a key role in the induction of mitosis bydephosphorylating and activating the PTK CDC2, leading to cell division(Sadhu, K. et al. (1990) Proc. Natl. Acad. Sci. USA 87:5139-5143). Inaddition, the genes encoding at least eight PTPs have been mapped tochromosomal regions that are translocated or rearranged in variousneoplastic conditions, including lymphoma, small cell lung carcinoma,leukemia, adenocarcinoma, and neuroblastoma (reviewed in Charbonneau, H.and N. K. Tonks (1992) Annu. Rev. Cell Biol. 8:463-493). The PTP enzymeactive site comprises the consensus sequence of the MTM1 gene family.The MTM1 gene is responsible for X-linked recessive myotubular myopathy,a congenital muscle disorder that has been linked to Xq28 (Kioschis, P.et al., (1998) Genomics 54:256-266). Many PTKs are encoded by oncogenes,and it is well known that oncogenesis is often accompanied by increasedtyrosine phosphorylation activity. It is therefore possible that PTPsmay serve to prevent or reverse cell transformation and the growth ofvarious cancers by controlling the levels of tyrosine phosphorylation incells. This is supported by studies showing that overexpression of PTPcan suppress transformation in cells and that specific inhibition of PTPcan enhance cell transformation (Charbonneau and Tonics, supra).

Apyrases are enzymes that efficiently hydrolyze ATP and ADP and mayfunction either intra- or extracellularly. One type of apyrase,ATP-diphosphohydrolase, catalyzes the hydrolysis of phosphoanhydridebonds of nucleoside tri- and di-phosphates in the presence of divalentcations (Nourizad, N. et al., (2003) Protein Purif. 27:229-237).

Dual specificity phosphatases (DSPs) are structurally more similar tothe PTPs than the PSPs. DSPs bear an extended PTP active site motif withan additional 7 amino acid residues. DSPs are primarily associated withcell proliferation and include the cell cycle regulators cdc25A, B, andC. The phosphatases DUSP1 and DUSP2 inactivate the MAPK family membersERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminalkinase), and p38 on both tyrosine and threonine residues (PROSITE PDOC00323, supra). In the activated state, these kinases have beenimplicated in neuronal differentiation, proliferation, oncogenictransformation, platelet aggregation, and apoptosis. Thus, DSPs arenecessary for proper regulation of these processes (Muda, M. et al.(1996) J. Biol. Chem. 271:27205-27208). The tumor suppressor PTEN is aDSP that also shows lipid phosphatase activity. It seems to negativelyregulate interactions with the extracellular matrix and maintainssensitivity to apoptosis. PTEN has been implicated in the prevention ofangiogenesis (Giri, D. and M. Ittmann (1999) Hum. Pathol. 30:419-424)and abnormalities in its expression are associated with numerous cancers(reviewed in Tamura, M. et al. (1999) J. Natl. Cancer Inst.91:1820-1828).

Histidine acid phosphatase (HAP; EXPASY EC 3.1.3.2), also known as acidphosphatase, hydrolyzes a wide spectrum of substrates including alkyl,aryl, and acyl orthophosphate monoesters and phosphorylated proteins atlow pH. HAPs share two regions of conserved sequences, each centeredaround a histidine residue which is involved in catalytic activity.Members of the HAP family include lysosomal acid phosphatase (LAP) andprostatic acid phosphatase (PAP), both sensitive to inhibition byL-tartrate (PROSITE PDOC00538).

Synaptojanin, a polyphosphoinositide phosphatase, dephosphorylatesphosphoinositides at positions 3, 4 and 5 of the inositol ring.Synaptojanin is a major presynaptic protein found at clathrin-coatedendocytic intermediates in nerve terminals, and binds the clathrincoat-associated protein, EPS15. This binding is mediated by theC-terminal region of synaptojanin-170, which has 3 Asp-Pro-Phe aminoacid repeats. Further, this 3 residue repeat had been found to be thebinding site for the EH domains of EPS15 (Haffner, C. et al. (1997) FEBSLett. 419:175-180). Additionally, synaptojanin may potentially regulateinteractions of endocytic proteins with the plasma membrane, and beinvolved in synaptic vesicle recycling (Brodin, L. et al. (2000) Curr.Opin. Neurobiol. 10:312-320). Studies in mice with a targeted disruptionin the synaptojanin 1 gene (Synj1) were shown to support coat formationof endocytic vesicles more effectively than was seen in wild-type mice,suggesting that Synj1 can act as a negative regulator of membrane-coatprotein interactions. These findings provide genetic evidence for acrucial role of phosphoinositide metabolism in synaptic vesiclerecycling (Cremona, O. et al. (1999) Cell 99:179-188).

Expression Profiling

Microarrays are analytical tools used in bioanalysis. A microarray has aplurality of molecules spatially distributed over, and stably associatedwith, the surface of a solid support. Microarrays of polypeptides,polynucleotides, and/or antibodies have been developed and find use in avariety of applications, such as gene sequencing, monitoring geneexpression, gene mapping, bacterial identification, drug discovery, andcombinatorial chemistry.

One area in particular in which microarrays find use is in geneexpression analysis. Array technology can provide a simple way toexplore the expression of a single polymorphic gene or the expressionprofile of a large number of related or unrelated genes. When theexpression of a single gene is examined, arrays are employed to detectthe expression of a specific gene or its variants. When an expressionprofile is examined, arrays provide a platform for identifying genesthat are tissue specific, are affected by a substance being tested in atoxicology assay, are part of a signaling cascade, carry outhousekeeping functions, or are specifically related to a particulargenetic predisposition, condition, disease, or disorder.

Neurological Disorders

Characterization of region-specific gene expression in the human brainprovides a context and background for molecular neurobiology on avariety of neurological disorders.

Alzheimer's disease (AD) is a progressive, neurodestructive process ofthe human neocortex, characterized by the deterioration of memory andhigher cognitive function. A progressive and irreversible braindisorder, AD is characterized by three major pathogenic episodesinvolving (a) an aberrant processing and deposition of beta-amyloidprecursor protein (betaAPP) to form neurotoxic beta-amyloid (betaA)peptides and an aggregated insoluble polymer of betaA that forms thesenile plaque, (b) the establishment of intraneuronal neuritic taupathology yielding widespread deposits of agyrophilic neurofibrillarytangles (NFT) and (c) the initiation and proliferation of abrain-specific inflammatory response. These three seemingly disperseattributes of AD etiopathogenesis are linked by the fact thatproinflammatory microglia, reactive astrocytes and their associatedcytokines and chemokines are associated with the biology of themicrotubule associated protein tau, betaA speciation and aggregation.Missense mutations in the presenilin genes PS1 and PS2, implicated inearly onset familial AD, cause abnormal betaAPP processing withresultant overproduction of betaA42 and related neurotoxic peptides.Specific betaA fragments such as betaA42 can further potentiateproinflammatory mechanisms. Expression of the inducible oxidoreductasecyclooxygenase-2 and cytosolic phospholipase A2 (cPLA2) is stronglyactivated during cerebral ischemia and trauma, epilepsy and AD,indicating the induction of proinflammatory gene pathways as a responseto brain injury. Neurotoxic metals such as aluminum and zinc, bothimplicated in AD etiopathogenesis, and arachidonic acid, a majormetabolite of brain cPLA2 activity, each polymerize hyperphosphorylatedtau to form NFT-like bundles. Studies have identified a reduced risk forAD in patients aged over 70 years who were previously treated withnon-steroidal anti-inflammatory drugs for non-CNS afflictions thatinclude arthritis. (For a review of the interrelationships between themechanisms of PS1, PS2 and betaAPP gene expression, tau and betaAdeposition and the induction, regulation and proliferation in AD of theneuroinflammatory response, see Lukiw, W. J, and Bazan, N. G. (2000)Neurochem. Res. 2000 25:1173-1184).

Breast Cancer

More than 180,000 new cases of breast cancer are diagnosed each year,and the mortality rate for breast cancer approaches 10% of all deaths infemales between the ages of 45-54 (Gish, K. (1999) AWIS Magazine28:7-10). However, the survival rate based on early diagnosis oflocalized breast cancer is extremely high (97%), compared with theadvanced stage of the disease in which the tumor has spread beyond thebreast (22%). Current procedures for clinical breast examination arelacking in sensitivity and specificity, and efforts are underway todevelop comprehensive gene expression profiles for breast cancer thatmay be used in conjunction with conventional screening methods toimprove diagnosis and prognosis of this disease (Perou, C. M. et al.(2000) Nature 406:747-752).

Mutations in two genes, BRCA1 and BRCA2, are known to greatly predisposea woman to breast cancer and may be passed on from parents to children(Gish, supra). However, this type of hereditary breast cancer accountsfor only about 5% to 9% of breast cancers, while the vast majority ofbreast cancer is due to non-inherited mutations that occur in breastepithelial cells.

The relationship between expression of epidermal growth factor (EGF) andits receptor, EGFR, to human mammary carcinoma has been particularlywell studied. (See Khazaie, K. et al. (1993) Cancer and Metastasis Rev.12:255-274, and references cited therein for a review of this area.)Overexpression of EGFR, particularly coupled with down-regulation of theestrogen receptor, is a marker of poor prognosis in breast cancerpatients. In addition, EGFR expression in breast tumor metastases isfrequently elevated relative to the primary tumor, suggesting that EGFRis involved in tumor progression and metastasis. This is supported byaccumulating evidence that EGF has effects on cell functions related tometastatic potential, such as cell motility, chemotaxis, secretion anddifferentiation. Changes in expression of other members of the erbBreceptor family, of which EGFR is one, have also been implicated inbreast cancer. The abundance of erbB receptors, such as HER-2/neu,HER-3, and HER-4, and their ligands in breast cancer points to theirfunctional importance in the pathogenesis of the disease, and maytherefore provide targets for therapy of the disease (Bacus, S. S. etal. (1994) Am. J. Clin. Pathol. 102:S13-S24). Other known markers ofbreast cancer include a human secreted frizzled protein mRNA that isdownregulated in breast tumors; the matrix G1a protein which isoverexpressed in human breast carcinoma cells; Drg1 or RTP, a gene whoseexpression is diminished in colon, breast, and prostate tumors; maspin,a tumor suppressor gene downregulated in invasive breast carcinomas; andCaN19, a member of the S100 protein family, all of which aredown-regulated in mammary carcinoma cells relative to normal mammaryepithelial cells (Zhou, Z. et al. (1998) Int. J. Cancer 78:95-99; Chen,L. et al. (1990) Oncogene 5:1391-1395; Ulrix, W. et a (1999) FEBS Lett455:23-26; Sager, R. et al. (1996) Curr. Top. Microbiol. Immunol.213:51-64; and Lee, S. W. et al. (1992) Proc. Natl. Acad. Sci. USA89:2504-2508).

Cell lines derived from human mammary epithelial cells at various stagesof breast cancer provide a useful model to study the process ofmalignant transformation and tumor progression as it has been shown thatthese cell lines retain many of the properties of their parental tumorsfor lengthy culture periods (Wistuba, I. I. et al. (1998) Clin. CancerRes. 4:2931-2938). Such a model is particularly useful for comparingphenotypic and molecular characteristics of human mammary epithelialcells at various stages of malignant transformation.

Colon Cancer

While soft tissue sarcomas are relatively rare, more than 50% of newpatients diagnosed with the disease will die from it. The molecularpathways leading to the development of sarcomas are relatively unknown,due to the rarity of the disease and variation in pathology. Coloncancer evolves through a multi-step process whereby pre-malignantcolonocytes undergo a relatively defined sequence of events leading totumor formation. Several factors participate in the process of tumorprogression and malignant transformation including genetic factors,mutations, and selection.

To understand the nature of gene alterations in colorectal cancer, anumber of studies have focused on the inherited syndromes. Familialadenomatous polyposis (FAP), is caused by mutations in the adenomatouspolyposis coli gene (APC), resulting in truncated or inactive forms ofthe protein. This tumor suppressor gene has been mapped to chromosome5q. Hereditary nonpolyposis colorectal cancer (HNPCC) is caused bymutations in mis-match repair genes. Although hereditary colon cancersyndromes occur in a small percentage of the population and mostcolorectal cancers are considered sporadic, knowledge from studies ofthe hereditary syndromes can be generally applied. For instance, somaticmutations in APC occur in at least 80% of sporadic colon tumors. APCmutations are thought to be the initiating event in the disease. Othermutations occur subsequently. Approximately 50% of colorectal cancerscontain activating mutations in ras, while 85% contain inactivatingmutations in p53. Changes in all of these genes lead to gene expressionchanges in colon cancer.

Lung Cancer

The potential application of gene expression profiling is particularlyrelevant to improving diagnosis, prognosis, and treatment of cancer,such as lung cancer. Lung cancer is the leading cause of cancer death inthe United States, affecting more than 100,000 men and 50,000 women eachyear. Nearly 90% of the patients diagnosed with lung cancer arecigarette smokers. Tobacco smoke contains thousands of noxioussubstances that induce carcinogen metabolizing enzymes and covalent DNAadduct formation in the exposed bronchial epithelium. In nearly 80% ofpatients diagnosed with lung cancer, metastasis has already occurred.Most commonly lung cancers metastasize to pleura, brain, bone,pericardium, and liver. The decision to treat with surgery, radiationtherapy, or chemotherapy is made on the basis of tumor histology,response to growth factors or hormones, and sensitivity to inhibitors ordrugs. With current treatments, most patients die within one year ofdiagnosis. Earlier diagnosis and a systematic approach toidentification, staging, and treatment of lung cancer could positivelyaffect patient outcome.

Lung cancers progress through a series of morphologically distinctstages from hyperplasia to invasive carcinoma. Malignant lung cancersare divided into two groups comprising four histopathological classes.The Non Small Cell Lung Carcinoma (NSCLC) group includes squamous cellcarcinomas, adenocarcinomas, and large cell carcinomas and accounts forabout 70% of all lung cancer cases. Adenocarcinomas typically arise inthe peripheral airways and often form mucin secreting glands. Squamouscell carcinomas typically arise in proximal airways. The histogenesis ofsquamous cell carcinomas may be related to chronic inflammation andinjury to the bronchial epithelium, leading to squamous metaplasia. TheSmall Cell Lung Carcinoma (SCLC) group accounts for about 20% of lungcancer cases. SCLCs typically arise in proximal airways and exhibit anumber of paraneoplastic syndromes including inappropriate production ofadrenocorticotropin and anti-diuretic hormone.

Lung cancer cells accumulate numerous genetic lesions, many of which areassociated with cytologically visible chromosomal aberrations. The highfrequency of chromosomal deletions associated with lung cancer mayreflect the role of multiple tumor suppressor loci in the etiology ofthis disease. Deletion of the short arm of chromosome 3 is found in over90% of cases and represents one of the earliest genetic lesions leadingto lung cancer. Deletions at chromosome arms 9p and 17p are also common.Other frequently observed genetic lesions include overexpression oftelomerase, activation of oncogenes such as K-ras and c-myc, andinactivation of tumor suppressor genes such as RB, p53 and CDKN2.

Genes differentially regulated in lung cancer have been identified by avariety of methods. Using mRNA differential display technology, Manda etal. (1999; Genomics 51:5-14) identified five genes differentiallyexpressed in lung cancer cell lines compared to normal bronchialepithelial cells. Among the known genes, pulmonary surfactant apoproteinA and alpha 2 macroglobulin were down regulated whereas nm23H1 wasupregulated. Petersen et al. (2000; Int J. Cancer, 86:512-517) usedsuppression subtractive hybridization to identify 552 clonesdifferentially expressed in lung tumor derived cell lines, 205 of whichrepresented known genes. Among the known genes, thrombospondin-1,fibronectin, intercellular adhesion molecule 1, and cytokeratins 6 and18 were previously observed to be differentially expressed in lungcancers. Wang et al. (2000; Oncogene 19:1519-1528) used a combination ofmicroarray analysis and subtractive hybridization to identify 17 genesdifferentially overexpressed in squamous cell carcinoma compared withnormal lung epithelium. Among the known genes they identified werekeratin isoform 6, KOC, SPRC, IGFb2, connexin 26, plakofillin 1 andcytokeratin 13.

Ovarian Cancer

Ovarian cancer is the leading cause of death from a gynecologic cancer.The majority of ovarian cancers are derived from epithelial cells, and70% of patients with epithelial ovarian cancers present with late-stagedisease. As a result, the long-term survival rate for this disease isvery low. Identification of early-stage markers for ovarian cancer wouldsignificantly increase the survival rate. Genetic variations involved inovarian cancer development include mutation of p53 and microsatelliteinstability. Gene expression patterns likely vary when normal ovary iscompared to ovarian tumors.

Prostate Cancer

As with most tumors, prostate cancer develops through a multistageprogression ultimately resulting in an aggressive tumor phenotype. Theinitial step in tumor progression involves the hyperproliferation ofnormal luminal and/or basal epithelial cells. Androgen responsive cellsbecome hyperplastic and evolve into early-stage tumors. Althoughearly-stage tumors are often androgen sensitive and respond to androgenablation, a population of androgen independent cells evolve from thehyperplastic population. These cells represent a more advanced form ofprostate tumor that may become invasive and potentially becomemetastatic to the bone, brain, or lung. A variety of genes may bedifferentially expressed during tumor progression. For example, loss ofheterozygosity (LOH) is frequently observed on chromosome 8p in prostatecancer. Fluorescence in situ hybridization (FISH) revealed a deletionfor at least 1 locus on 8p in 29 (69%) tumors, with a significantlyhigher frequency of the deletion on 8p21.2-p21.1 in advanced prostatecancer than in localized prostate cancer, implying that deletions on8p22-p21.3 play an important role in tumor differentiation, while8p21.2-p21.1 deletion plays a role in progression of prostate cancer(Oba, K. et al. (2001) Cancer Genet. Cytogenet. 124: 20-26).

PZ-HPV-7 was derived from epithelial cells cultured from normal tissuefrom the peripheral zone of the prostate. The cells were transformed bytransfection with HPV18. Immunocytochemical analysis showed expressionof keratins 5 and 8 and also the early region 6 (E6) oncoprotein of HPV.The cells are negative for prostate specific antigen (PSA).

Interleukin 6 (IL-6) is a multifunctional protein that plays importantroles in host defense, acute phase reactions, immune responses, andhematopoiesis. According to the type of biological responses beingstudied, IL-6 was previously named interferon-b2, 26-kDa protein, B cellstimulatory factor-2 (BSF-2), hybridoma/plasmacytoma growth factor,hepatocyte stimulating factor, cytotoxic T cell differentiation factor,and macrophage-granulocyte inducing factor 2A (MGI-2A). The IL-6designation was adopted after these variously named proteins were foundto be identical on the basis of their amino acid and/or nucleotidesequences. IL-6 is expressed by a variety of normal and transformedcells including T cells, B cells, monocytes/macrophages, fibroblasts,hepatocytes, keratinocytes, astrocytes, vascular endothelial cells, andvarious tumor cells. The production of IL-6 is upregulated by numeroussignals including mitogenic or antigenic stimulation, LPS, calciumionophore, IL-1, IL-2, IFN, TNF, PDGF, and viruses. IL-4 and IL-13inhibit IL-6 expression in monocytes.

Obesity

The most important function of adipose tissue is its ability to storeand release fat during periods of feeding and fasting. White adiposetissue is the major energy reserve in periods of excess energy use. Itsprimary purpose is mobilization during energy deprivation. Understandinghow various molecules regulate adiposity and energy balance inphysiological and pathophysiological situations may lead to thedevelopment of novel therapeutics for human obesity. Adipose tissue isalso one of the important target tissues for insulin. Adipogenesis andinsulin resistance in type II diabetes are linked and present intriguingrelations. Most patients with type II diabetes are obese and obesity inturn causes insulin resistance.

The majority of research in adipocyte biology to date has been doneusing transformed mouse preadipocyte cell lines. The culture conditionwhich stimulates mouse preadipocyte differentiation is different fromthat for inducing human primary preadipocyte differentiation. Inaddition, primary cells are diploid and may therefore reflect the invivo context better than aneuploid cell lines. Understanding the geneexpression profile during adipogenesis in humans will lead to anunderstanding of the fundamental mechanism of adiposity regulation.Furthermore, through comparing the gene expression profiles ofadipogenesis between donors with normal weight and donors with obesity,identification of crucial genes, potential drug targets for obesity andtype II diabetes, will be possible.

Thiazolidinediones (TZDs) act as agonists for theperoxisome-proliferator-activated receptor gamma (PPARγ), a member ofthe nuclear hormone receptor superfamily. TZDs reduce hyperglycemia,hyperinsulinemia, and hypertension, in part by promoting glucosemetabolism and inhibiting gluconeogenesis. Roles for PPARγ and itsagonists have been demonstrated in a wide range of pathologicalconditions including diabetes, obesity, hypertension, atherosclerosis,polycystic ovarian syndrome, and cancers such as breast, prostate,liposarcoma, and colon cancer.

The mechanism by which TZDs and other PPARγ agonists enhance insulinsensitivity is not fully understood, but may involve the ability ofPPARγ to promote adipogenesis. When ectopically expressed in culturedpreadipocytes, PPARγ is a potent inducer of adipocyte differentiation.TZDs, in combination with insulin and other factors, can also enhancedifferentiation of human preadipocytes in culture (Adams et al. (1997)J. Clin. Invest. 100:3149-3153). The relative potency of different TZDsin promoting adipogenesis in vitro is proportional to both their insulinsensitizing effects in vivo, and their ability to bind and activatePPARγ in vitro. Interestingly, adipocytes derived from omental adiposedepots are refractory to the effects of TZDs. It has therefore beensuggested that the insulin sensitizing effects of TZDs may result fromtheir ability to promote adipogenesis in subcutaneous adipose depots(Adams et al., supra). Further, dominant negative mutations in the PPARγgene have been identified in two non-obese subjects with severe insulinresistance, hypertension, and overt non-insulin dependent diabetesmellitus (NIDDM) (Barroso et al. (1998) Nature 402:880-883).

NIDDM is the most common form of diabetes mellitus, a chronic metabolicdisease that affects 143 million people worldwide. NIDDM ischaracterized by abnormal glucose and lipid metabolism that results froma combination of peripheral insulin resistance and defective insulinsecretion. NIDDM has a complex, progressive etiology and a high degreeof heritability. Numerous complications of diabetes including heartdisease, stroke, renal failure, retinopathy, and peripheral neuropathycontribute to the high rate of morbidity and mortality.

At the molecular level, PPARγ functions as a ligand activatedtranscription factor. In the presence of ligand, PPARγ forms aheterodimer with the retinoid X receptor (RXR) which then activatestranscription of target genes containing one or more copies of a PPARγresponse element (PPRE). Many genes important in lipid storage andmetabolism contain PPREs and have been identified as PPARγ targets,including PEPCK, aP2, LPL, ACS, and FAT-P (Auwerx, J. (1999)Diabetologia 42:1033-1049). Multiple ligands for PPARγ have beenidentified. These include a variety of fatty acid metabolites; syntheticdrugs belonging to the TZD class, such as Pioglitazone and Rosiglitazone(BRL49653); and certain non-glitazone tyrosine analogs such as GI262570and GW1929. The prostaglandin derivative 15-dPGJ2 is a potent endogenousligand for PPARγ.

Expression of PPARγ is very high in adipose but barely detectable inskeletal muscle, the primary site for insulin stimulated glucosedisposal in the body. PPARγ is also moderately expressed in largeintestine, kidney, liver, vascular smooth muscle, hematopoietic cells,and macrophages. The high expression of PPARγ in adipose tissue suggeststhat the insulin sensitizing effects of TZDs may result from alterationsin the expression of one or more PPARγ regulated genes in adiposetissue. Identification of PPARγ target genes will contribute to betterdrug design and the development of novel therapeutic strategies fordiabetes, obesity, and other conditions.

Systematic attempts to identify PPARγ target genes have been made inseveral rodent models of obesity and diabetes (Suzuki et al. (2000) Jpn.J. Pharmacol. 84:113-123; Way et al. (2001) Endocrinology142:1269-1277). However, a serious drawback of the rodent geneexpression studies is that significant differences exist between humanand rodent models of adipogenesis, diabetes, and obesity (Taylor (1999)Cell 97:9-12; Gregoire et al. (1998) Physiol. Reviews 78:783-809).Therefore, an unbiased approach to identifying TZD regulated genes inprimary cultures of human tissues is necessary to fully elucidate themolecular basis for diseases associated with PPARγ activity.

There is a need in the art for new compositions, including nucleic acidsand proteins, for the diagnosis, prevention, and treatment ofcardiovascular diseases, immune system disorders, neurologicaldisorders, disorders affecting growth and development, lipid disorders,cell proliferative disorders, and cancers.

SUMMARY OF THE INVENTION

Various embodiments of the invention provide purified polypeptides,kinases and phosphatases, referred to collectively as ‘KPP’ andindividually as ‘KPP-1,’ ‘KPP-2,’ ‘KPP-3,’ ‘KPP-4,’ ‘KPP-5,’ ‘KPP-6,’‘KPP-7,’ ‘KPP-8,’ ‘KPP-9,’ ‘KPP-10,’ ‘KPP-11,’ ‘KPP-12,’ ‘KPP-13,’‘KPP-14,’ ‘KPP-15,’ ‘KPP-16,’ ‘KPP-17,’ ‘KPP-18,’ ‘KPP-19,’ ‘KPP-20,’‘KPP-21,’ ‘KPP-22,’ ‘KPP-23’, ‘KPP-24,’ ‘KPP-25,’ ‘KPP-26,’ ‘KPP-27,’‘KPP-28,’ ‘KPP-29,’ ‘KPP-30,’ ‘KPP-31,’ ‘KPP-32,’ ‘KPP-33,’ ‘KPP-34,’‘KPP-35,’ ‘KPP-36,’ ‘KPP-37,’ ‘KPP-38,’ ‘KPP-39,’ ‘KPP-40,’ ‘KPP-41,’‘KPP-42,’ and ‘KPP-43’ and methods for using these proteins and theirencoding polynucleotides for the detection, diagnosis, and treatment ofdiseases and medical conditions Embodiments also provide methods forutilizing the purified kinases and phosphatases and/or their encodingpolynucleotides for facilitating the drug discovery process, includingdetermination of efficacy, dosage, toxicity, and pharmacology. Relatedembodiments provide methods for utilizing the purified kinases andphosphatases and/or their encoding polynucleotides for investigating thepathogenesis of diseases and medical conditions.

An embodiment provides an isolated polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-43, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-43, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-43, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-43. Another embodiment provides anisolated polypeptide comprising an amino acid sequence of SEQ IDNO:1-43.

Still another embodiment provides an isolated polynucleotide encoding apolypeptide selected from the group consisting of a) a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-43, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical or at least about 90% identical toan amino acid sequence selected from the group consisting of SEQ IDNO:1-43, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-43, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-43. Inanother embodiment, the polynucleotide encodes a polypeptide selectedfrom the group consisting of SEQ ID NO:1-43. In an alternativeembodiment, the polynucleotide is selected from the group consisting ofSEQ ID NO:44-86.

Still another embodiment provides a recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotideencoding a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical or at least about90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43. Another embodiment provides a celltransformed with the recombinant polynucleotide. Yet another embodimentprovides a transgenic organism comprising the recombinantpolynucleotide.

Another embodiment provides a method for producing a polypeptideselected from the group consisting of a) a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ IDNO:1-43, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical or at least about 90% identical to anamino acid sequence selected from the group consisting of SEQ IDNO:1-43, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-43, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-43. Themethod comprises a) culturing a cell under conditions suitable forexpression of the polypeptide, wherein said cell is transformed with arecombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide encoding the polypeptide, and b) recoveringthe polypeptide so expressed.

Yet another embodiment provides an isolated antibody which specificallybinds to a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical or at least about90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43.

Still yet another embodiment provides an isolated polynucleotideselected from the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:44-86, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:44-86, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). In otherembodiments, the polynucleotide can comprise at least about 20, 30, 40,60, 80, or 100 contiguous nucleotides.

Yet another embodiment provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide being selectedfrom the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:44-86, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:44-86, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). The methodcomprises a) hybridizing the sample with a probe comprising at least 20contiguous nucleotides comprising a sequence complementary to saidtarget polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex. In a related embodiment, themethod can include detecting the amount of the hybridization complex. Instill other embodiments, the probe can comprise at least about 20, 30,40, 60, 80, or 100 contiguous nucleotides.

Still yet another embodiment provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide being selectedfrom the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:44-86, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:44-86, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). The methodcomprises a) amplifying said target polynucleotide or fragment thereofusing polymerase chain reaction amplification, and b) detecting thepresence or absence of said amplified target polynucleotide or fragmentthereof. In a related embodiment, the method can include detecting theamount of the amplified target polynucleotide or fragment thereof.

Another embodiment provides a composition comprising an effective amountof a polypeptide selected from the group consisting of a) a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-43, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical or at least about 90% identical toan amino acid sequence selected from the group consisting of SEQ IDNO:1-43, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-43, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-43, anda pharmaceutically acceptable excipient In one embodiment, thecomposition can comprise an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43. Other embodiments provide a method oftreating a disease or condition associated with decreased or abnormalexpression of functional KPP, comprising administering to a patient inneed of such treatment the composition.

Yet another embodiment provides a method for screening a compound foreffectiveness as an agonist of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-43, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-43, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-43, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-43. The method comprises a)contacting a sample comprising the polypeptide with a compound, and b)detecting agonist activity in the sample. Another embodiment provides acomposition comprising an agonist compound identified by the method anda pharmaceutically acceptable excipient. Yet another embodiment providesa method of treating a disease or condition associated with decreasedexpression of functional KPP, comprising administering to a patient inneed of such treatment the composition.

Still yet another embodiment provides a method for screening a compoundfor effectiveness as an antagonist of a polypeptide selected from thegroup consisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-43, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-43, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-43, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-43. The method comprises a)contacting a sample comprising the polypeptide with a compound, and b)detecting antagonist activity in the sample. Another embodiment providesa composition comprising an antagonist compound identified by the methodand a pharmaceutically acceptable excipient. Yet another embodimentprovides a method of treating a disease or condition associated withoverexpression of functional KPP, comprising administering to a patientin need of such treatment the composition.

Another embodiment provides a method of screening for a compound thatspecifically binds to a polypeptide selected from the group consistingof a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-43, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical or atleast about 90% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-43, c) a biologically active fragment ofa polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-43. The method comprises a) combining thepolypeptide with at least one test compound under suitable conditions,and b) detecting binding of the polypeptide to the test compound,thereby identifying a compound that specifically binds to thepolypeptide.

Yet another embodiment provides a method of screening for a compoundthat modulates the activity of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-43, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-43, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-43, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-43. The method comprises a)combining the polypeptide with at least one test compound underconditions permissive for the activity of the polypeptide, b) assessingthe activity of the polypeptide in the presence of the test compound;and c) comparing the activity of the polypeptide in the presence of thetest compound with the activity of the polypeptide in the absence of thetest compound, wherein a change in the activity of the polypeptide inthe presence of the test compound is indicative of a compound thatmodulates the activity of the polypeptide.

Still yet another embodiment provides a method for screening a compoundfor effectiveness in altering expression of a target polynucleotide,wherein said target polynucleotide comprises a polynucleotide sequenceselected from the group consisting of SEQ ID NO:44-86, the methodcomprising a) contacting a sample comprising the target polynucleotidewith a compound, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.

Another embodiment provides a method for assessing toxicity of a testcompound, said method comprising a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide selected from thegroup consisting of i) a polynucleotide comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:44-86, ii) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical or at least about 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:44-86, iii) a polynucleotide having a sequence complementary to i),iv) a polynucleotide complementary to the polynucleotide of ii), and v)an RNA equivalent of i)-iv). Hybridization occurs under conditionswhereby a specific hybridization complex is formed between said probeand a target polynucleotide in the biological sample, said targetpolynucleotide selected from the group consisting of i) a polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO:44-86, ii) a polynucleotide comprising a naturallyoccurring polynucleotide sequence at least 90% identical or at leastabout 90% identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:44-86, iii) a polynucleotide complementary tothe polynucleotide of i), iv) a polynucleotide complementary to thepolynucleotide of and v) an RNA equivalent of i)-iv). Alternatively, thetarget polynucleotide can comprise a fragment of a polynucleotideselected from the group consisting of i)-v) above; c) quantifying theamount of hybridization complex; and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for full length polynucleotide andpolypeptide embodiments of the invention.

Table 2 shows the GenBank identification number and annotation of thenearest GenBank homolog, and the PROTEOME database identificationnumbers and annotations of PROTEOME database homologs, for polypeptideembodiments of the invention. The probability scores for the matchesbetween each polypeptide and its homologs) are also shown.

Table 3 shows structural features of polypeptide embodiments, includingpredicted motifs and domains, along with the methods, algorithms, andsearchable databases used for analysis of the polypeptides.

Table 4 lists the cDNA and/or genomic DNA fragments which were used toassemble polynucleotide embodiments, along with selected fragments ofthe polynucleotides.

Table 5 shows representative cDNA libraries for polynucleotideembodiments.

Table 6 provides an appendix which describes the tissues and vectorsused for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyzepolynucleotides and polypeptides, along with applicable descriptions,references, and threshold parameters.

Table 8 shows single nucleotide polymorphisms found in polynucleotidesequences of the invention, along with allele frequencies in differenthuman populations.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleic acids, and methods are described,it is understood that embodiments of the invention are not limited tothe particular machines, instruments, materials, and methods described,as these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a host cell” includes aplurality of such host cells, and a reference to “an antibody” is areference to one or more antibodies and equivalents thereof known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with variousembodiments of the invention. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention:

DEFINITIONS

“KPP” refers to the amino acid sequences of substantially purified KPPobtained from any species, particularly a mammalian species, includingbovine, ovine, porcine, murine, equine, and human, and from any source,whether natural, synthetic, semi-synthetic, or recombinant.

The term “agonist” refers to a molecule which intensifies or mimics thebiological activity of KPP. Agonists may include proteins, nucleicacids, carbohydrates, small molecules, or any other compound orcomposition which modulates the activity of KPP either by directlyinteracting with KPP or by acting on components of the biologicalpathway in which KPP participates.

An “allelic variant” is an alternative form of the gene encoding KPP.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. A gene may have none,one, or many allelic variants of its naturally occurring form. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding KPP include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polypeptide the same as KPP or a polypeptide with atleast one functional characteristic of KPP. Included within thisdefinition are polymorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingKPP, and improper or unexpected hybridization to allelic variants, witha locus other than the normal chromosomal locus for the polynucleotideencoding KPP. The encoded protein may also be “altered,” and may containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent KPP.Deliberate amino acid substitutions may be made on the basis of one ormore similarities in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas the biological or immunological activity of KPP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid, and positively charged amino acids may include lysine andarginine. Amino acids with uncharged polar side chains having similarhydrophilicity values may include: asparagine and glutamine; and serineand threonine. Amino acids with uncharged side chains having similarhydrophilicity values may include: leucine, isoleucine, and valine;glycine and alanine; and phenylalanine and tyrosine.

The terms “amino acid” and “amino acid sequence” can refer to anoligopeptide, a peptide, a polypeptide, or a protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. Where “amino acid sequence” is recited to refer to a sequenceof a naturally occurring protein molecule, “amino acid sequence” andlike terms are not meant to limit the amino acid sequence to thecomplete native amino acid sequence associated with the recited proteinmolecule.

“Amplification” relates to the production of additional copies of anucleic acid. Amplification may be carried out using polymerase chainreaction (PCR) technologies or other nucleic acid amplificationtechnologies well known in the art.

The term “antagonist” refers to a molecule which inhibits or attenuatesthe biological activity of KPP. Antagonists may include proteins such asantibodies, anticalins, nucleic acids, carbohydrates, small molecules,or any other compound or composition which modulates the activity of KPPeither by directly interacting with KPP or by acting on components ofthe biological pathway in which KPP participates.

The term “antibody” refers to intact immunoglobulin molecules as well asto fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding an epitopic determinant. Antibodies that bind KPPpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant” refers to that region of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (particular regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune-response) for binding to an antibody.

The term “aptamer” refers to a nucleic acid or oligonucleotide moleculethat binds to a specific molecular target. Aptamers are derived from anin vitro evolutionary process (e.g., SELEX (Systematic Evolution ofLigands by EXponential Enrichment), described in U.S. Pat. No.5,270,163), which selects for target-specific aptamer sequences fromlarge combinatorial libraries. Aptamer compositions may bedouble-stranded or single-stranded, and may includedeoxyribonucleotides, ribonucleotides, nucleotide derivatives, or othernucleotide-like molecules. The nucleotide components of an aptamer mayhave modified sugar groups (e.g., the 2′-OH group of a ribonucleotidemay be replaced by 2′-F or 2′—NH₂), which may improve a desiredproperty, e.g., resistance to nucleases or longer lifetime in blood.Aptamars may be conjugated to other molecules, e.g., a high molecularweight carrier to slow clearance of the aptamer from the circulatorysystem. Aptamers may be specifically cross-linked to their cognateligands, e.g., by photo-activation of a cross-linker (Brody, E. N. andL. Gold (2000) J. Biotechnol. 74:5-13).

The term “intramer” refers to an aptamer which is expressed in vivo. Forexample, a vaccinia virus-based RNA expression system has been used toexpress specific RNA aptamers at high levels in the cytoplasm ofleukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA96:3606-3610).

The term “spiegelmer” refers to an aptamer which includes L-DNA, L-RNA,or other left-handed nucleotide derivatives or nucleotide-likemolecules. Aptamers containing left-handed nucleotides are resistant todegradation by naturally occurring enzymes, which normally act onsubstrates containing right-handed nucleotides.

The term “antisense” refers to any composition capable of base-pairingwith the “sense” (coding) strand of a polynucleotide having a specificnucleic acid sequence. Antisense compositions may include DNA; RNA;peptide nucleic acid (PNA); oligonucleotides having modified backbonelinkages such as phosphorothioates, methylphosphonates, orbenzylphosphonates; oligonucleotides having modified sugar groups suchas 2′-methoxyethyl sugars or 2′-methoxyethoxy sugars; oroligonucleotides having modified bases such as 5-methyl cytosine,2′-deoxyuracil, or 7-deaza-2′-deoxyguanosine. Antisense molecules may beproduced by any method including chemical synthesis or transcription.Once introduced into a cell, the complementary antisense molecule basepairs with a naturally occurring nucleic acid sequence produced by thecell to form duplexes which block either transcription or translation.The designation “negative” or “minus” can refer to the antisense strand,and the designation “positive” or “plus” can refer to the sense strandof a reference DNA molecule.

The term “biologically active” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” or “immunogenic” refers to thecapability of the natural, recombinant, or synthetic KPP, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

“Complementary” describes the relationship between two single-strandednucleic acid sequences that anneal by base-pairing. For example,5′-AGT-3′ pairs with its complement, 3′-TCA-5′.

A “composition comprising a given polynucleotide” and a “compositioncomprising a given polypeptide” can refer to any composition containingthe given polynucleotide or polypeptide. The composition may comprise adry formulation or an aqueous solution. Compositions comprisingpolynucleotides encoding KPP or fragments of KPP may be employed ashybridization probes. The probes may be stored in freeze-dried form andmay be associated with a stabilizing agent such as a carbohydrate. Inhybridizations, the probe may be deployed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate;SDS), and other components (e.g., Denhardt's solution, dry milk, salmonsperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beensubjected to repeated DNA sequence analysis to resolve uncalled bases,extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.)in the 5′ and/or the 3′ direction, and resequenced, or which has beenassembled from one or more overlapping cDNA, EST, or genomic DNAfragments using a computer program for fragment assembly, such as theGELVIEW fragment assembly system (Accelrys, Burlington Mass.) or Phrap(University of Washington, Seattle Wash.). Some sequences have been bothextended and assembled to produce the consensus sequence.

“Conservative amino acid substitutions” are those substitutions that arepredicted to least interfere with the properties of the originalprotein, i.e., the structure and especially the function of the proteinis conserved and not significantly changed by such substitutions. Thetable below shows amino acids which may be substituted for an originalamino acid in a protein and which are regarded as conservative aminoacid substitutions.

Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys AsnAsp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln,His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg,Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser,Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

Conservative amino acid substitutions generally maintain (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a beta sheet or alpha helical conformation, (b) thecharge or hydrophobicity of the molecule at the site of thesubstitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to a chemically modified polynucleotide orpolypeptide. Chemical modifications of a polynucleotide can include, forexample, replacement of hydrogen by an alkyl, acyl, hydroxyl, or aminogroup. A derivative polynucleotide encodes a polypeptide which retainsat least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

A “detectable label” refers to a reporter molecule or enzyme that iscapable of generating a measurable signal and is covalently ornoncovalently joined to a polynucleotide or polypeptide.

“Differential expression” refers to increased or upregulated; ordecreased, downregulated, or absent gene or protein expression,determined by comparing at least two different samples. Such comparisonsmay be carried out between, for example, a treated and an untreatedsample, or a diseased and a normal sample.

“Exon shuffling” refers to the recombination of different coding regions(exons). Since an exon may represent a structural or functional domainof the encoded protein, new proteins may be assembled through the novelreassortment of stable substructures, thus allowing acceleration of theevolution of new protein functions.

A “fragment” is a unique portion of KPP or a polynucleotide encoding KPPwhich can be identical in sequence to, but shorter in length than, theparent sequence. A fragment may comprise up to the entire length of thedefined sequence, minus one nucleotide/amino acid residue. For example,a fragment may comprise from about 5 to about 1000 contiguousnucleotides or amino acid residues. A fragment used as a probe, primer,antigen, therapeutic molecule, or for other purposes, may be at least 5,10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500contiguous nucleotides or amino acid residues in length. Fragments maybe preferentially selected from certain regions of a molecule. Forexample, a polypeptide fragment may comprise a certain length ofcontiguous amino acids selected from the first 250 or 500 amino acids(or first 25% or 50%) of a polypeptide as shown in a certain definedsequence. Clearly these lengths are exemplary, and any length that issupported by the specification, including the Sequence Listing, tables,and figures, may be encompassed by the present embodiments.

A fragment of SEQ ID NO:44-86 can comprise a region of uniquepolynucleotide sequence that specifically identifies SEQ ID NO:44-86,for example, as distinct from any other sequence in the genome fromwhich the fragment was obtained. A fragment of SEQ ID NO:44-86 can beemployed in one or more embodiments of methods of the invention, forexample, in hybridization and amplification technologies and inanalogous methods that distinguish SEQ ID NO:44-86 from relatedpolynucleotides. The precise length of a fragment of SEQ ID NO:44-86 andthe region of SEQ ID NO:44-86 to which the fragment corresponds areroutinely determinable by one of ordinary skill in the art based on theintended purpose for the fragment.

A fragment of SEQ ID NO:1-43 is encoded by a fragment of SEQ IDNO:44-86. A fragment of SEQ ID NO:1-43 can comprise a region of uniqueamino acid sequence that specifically identifies SEQ ID NO:1-43. Forexample, a fragment of SEQ ID NO:1-43 can be used as an immunogenicpeptide for the development of antibodies that specifically recognizeSEQ ID NO:1-43. The precise length of a fragment of SEQ ID NO:1-43 andthe region of SEQ ID NO:1-43 to which the fragment corresponds can bedetermined based on the intended purpose for the fragment using one ormore analytical methods described herein or otherwise known in the art.

A “full length” polynucleotide is one containing at least a translationinitiation codon (e.g., methionine) followed by an open reading frameand a translation termination codon. A “full length” polynucleotidesequence encodes a “full length” polypeptide sequence.

“Homology” refers to sequence similarity or, alternatively, sequenceidentity, between two or more polynucleotide sequences or two or morepolypeptide sequences.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of identicalnucleotide matches between at least two polynucleotide sequences alignedusing a standardized algorithm. Such an algorithm may insert, in astandardized and reproducible way, gaps in the sequences being comparedin order to optimize alignment between two sequences, and thereforeachieve a more meaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determinedusing one or more computer algorithms or programs known in the art ordescribed herein. For example, percent identity can be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program. This program ispart of the LASERGENE software package, a suite of molecular biologicalanalysis programs (DNASTAR, Madison Wis.). CLUSTAL V is described inHiggins, D. G. and P. M. Sharp (1989; CABIOS 5:151-153) and in Higgins,D. G. et al. (1992; CABIOS 8:189-191). For pairwise alignments ofpolynucleotide sequences, the default parameters are set as follows:Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4. The“weighted” residue weight table is selected as the default.

Alternatively, a suite of commonly used and freely available sequencecomparison algorithms which can be used is provided by the NationalCenter for Biotechnology Information (NCBI) Basic Local Alignment SearchTool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410),which is available from several sources, including the NCBI, Bethesda,Md., and on the Internet at ncbi.nlm.nih.gov/BLAST/. The BLAST softwaresuite includes various sequence analysis programs including “blastn,”that is used to align a known polynucleotide sequence with otherpolynucleotide sequences from a variety of databases. Also available isa tool called “BLAST 2 Sequences” that is used for direct pairwisecomparison of two nucleotide sequences. “BLAST 2 Sequences” can beaccessed and used interactively at ncbi.nlm.nih.gov/gorf/b12.html. The“BLAST 2 Sequences” tool can be used for both blastn and blastp(discussed below). BLAST programs are commonly used with gap and otherparameters set to default settings. For example, to compare twonucleotide sequences, one may use blastn with the “BLAST 2 Sequences”tool Version 2.0.12 (April-21-2000) set at default parameters. Suchdefault parameters may be, for example:

Matrix: BLOSUM62

Reward for match: 1

Penalty for mismatch: −2

Open Gap: 5 and Extension Gap: 2 penalties

Gap×drop-off. 50

Expect: 10

Word Size: 11

Filter: on

Percent identity may be measured over the length of an entire definedsequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of afragment taken from a larger, defined sequence, for instance, a fragmentof at least 20, at least 30, at least 40, at least 50, at least 70, atleast 100, or at least 200 contiguous nucleotides. Such lengths areexemplary only, and it is understood that any fragment length supportedby the sequences shown herein, in the tables, figures, or SequenceListing, may be used to describe a length over which percentage identitymay be measured.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof the genetic code. It is understood that changes in a nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of identical residuematches between at least two polypeptide sequences aligned using astandardized algorithm. Methods of polypeptide sequence alignment arewell-known. Some alignment methods take into account conservative aminoacid substitutions. Such conservative substitutions, explained in moredetail above, generally preserve the charge and hydrophobicity at thesite of substitution, thus preserving the structure (and thereforefunction) of the polypeptide. The phrases “percent similarity” and “%similarity,” as applied to polypeptide sequences, refer to thepercentage of residue matches, including identical residue matches andconservative substitutions, between at least two polypeptide sequencesaligned using a standardized algorithm. In contrast, conservativesubstitutions are not included in the calculation of percent identitybetween polypeptide sequences.

Percent identity between polypeptide sequences may be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program (described andreferenced above). For pairwise alignments of polypeptide sequencesusing CLUSTAL V, the default parameters are set as follows: Ktuple=1,gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix isselected as the default residue weight table.

Alternatively the NCBI BLAST software suite may be used. For example,for a pairwise comparison of two polypeptide sequences, one may use the“BLAST 2 Sequences” tool Version 2.0.12 (April-21-2000) with blastp setat default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Open Gap: 11 and Extension Gap: 1 penalties

Gap×drop-off. 50

Expect: 10

Word Size: 3

Filter: on

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size and whichcontain all of the elements required for chromosome replication,segregation and maintenance.

The term “humanized antibody” refers to an antibody molecule in whichthe amino acid sequence in the non-antigen binding regions has beenaltered so that the antibody more closely resembles a human antibody,and still retains its original binding ability.

“Hybridization” refers to the process by which a polynucleotide strandanneals with a complementary strand through base pairing under definedhybridization conditions. Specific hybridization is an indication thattwo nucleic acid sequences share a high degree of complementarity.Specific hybridization complexes form under permissive annealingconditions and remain hybridized after the “washing” step(s). Thewashing step(s) is particularly important in determining the stringencyof the hybridization process, with more stringent conditions allowingless non-specific binding, i.e., binding between pairs of nucleic acidstrands that are not perfectly matched. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may be consistent among hybridizationexperiments, whereas wash conditions may be varied among experiments toachieve the desired stringency, and therefore hybridization specificity.Permissive annealing conditions occur, for example, at 68° C. in thepresence of about 6×SSC, about 1% (w/v) SDS, and about 100 μg/mlsheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, withreference to the temperature under which the wash step is carried out.Such wash temperatures are typically selected to be about 5° C. to 20°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. An equation forcalculating T_(m) and conditions for nucleic acid hybridization are wellknown and can be found in Sambrook, J. and D. W. Russell (2001;Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold SpringHarbor Press, Cold Spring Harbor N.Y., ch. 9).

High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68° C. in the presenceof about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively,temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSCconcentration may be varied from about 0.1 to 2×SSC, with SDS beingpresent at about 0.1%. Typically, blocking reagents are used to blocknon-specific hybridization. Such blocking reagents include, forinstance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml.Organic solvent, such as formamide at a concentration of about 35-50%v/v, may also be used under particular circumstances, such as forRNA:DNA hybridizations. Useful variations on these wash conditions willbe readily apparent to those of ordinary skill in the art.Hybridization, particularly under high stringency conditions, may besuggestive of evolutionary similarity between the nucleotides. Suchsimilarity is strongly indicative of a similar role for the nucleotidesand their encoded polypeptides.

The term “hybridization complex” refers to a complex formed between twonucleic acids by virtue of the formation of hydrogen bonds betweencomplementary bases. A hybridization complex may be formed in solution(e.g., C₀t or R₀t analysis) or formed between one nucleic acid presentin solution and another nucleic acid immobilized on a solid support(e.g., paper, membranes, filters, chips, pins or glass slides, or anyother appropriate substrate to which cells or their nucleic acids havebeen fixed).

The words “insertion” and “addition” refer to changes in an amino acidor polynucleotide sequence resulting in the addition of one or moreamino acid residues or nucleotides, respectively.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

An “immunogenic fragment” is a polypeptide or oligopeptide fragment ofKPP which is capable of eliciting an immune response when introducedinto a living organism, for example, a mammal. The term “immunogenicfragment” also includes any polypeptide or oligopeptide fragment of KPPwhich is useful in any of the antibody production methods disclosedherein or known in the art.

The term “microarray” refers to an arrangement of a plurality ofpolynucleotides, polypeptides, antibodies, or other chemical compoundson a substrate.

The terms “element” and “array element” refer to a polynucleotide,polypeptide, antibody, or other chemical compound having a unique anddefined position on a microarray.

The term “modulate” refers to a change in the activity of KPP. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of KPP.

The phrases “nucleic acid” and “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material.

“Operably linked” refers to the situation in which a first nucleic acidsequence is placed in a functional relationship with a second nucleicacid sequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Operably linked DNA sequences may be in close proximityor contiguous and, where necessary to join two protein coding regions,in the same reading frame.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

“Post-translational modification” of an KPP may involve lipidation,glycosylation, phosphorylation, acetylation, racemization, proteolyticcleavage, and other modifications known in the art. These processes mayoccur synthetically or biochemically. Biochemical modifications willvary by cell type depending on the enzymatic milieu of KPP.

“Probe” refers to nucleic acids encoding KPP, their complements, orfragments thereof, which are used to detect identical, allelic orrelated nucleic acids. Probes are isolated oligonucleotides orpolynucleotides attached to a detectable label or reporter molecule.Typical labels include radioactive isotopes, ligands, chemiluminescentagents, and enzymes. “Primers” are short nucleic acids, usually DNAoligonucleotides, which may be annealed to a target polynucleotide bycomplementary base-pairing. The primer may then be extended along thetarget DNA strand by a DNA polymerase enzyme. Primer pairs can be usedfor amplification (and identification) of a nucleic acid, e.g., by thepolymerase chain reaction (PCR).

Probes and primers as used in the present invention typically compriseat least 15 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed,such as probes and primers that comprise at least 20, 25, 30, 40, 50,60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of thedisclosed nucleic acid sequences. Probes and primers may be considerablylonger than these examples, and it is understood that any lengthsupported by the specification, including the tables, figures, andSequence Listing, may be used.

Methods for preparing and using probes and primers are described in, forexample, Sambrook, J. and D. W. Russell (2001; Molecular Cloning: ALaboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, ColdSpring Harbor N.Y.), Ausubel, F. M. et al. (1999; Short Protocols inMolecular Biology, 4^(th) ed., John Wiley & Sons, New York N.Y.), andInnis, M. et al. (1990; PCR Protocols, A Guide to Methods andApplications, Academic Press, San Diego Calif.). PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers are selected using software known inthe art for such purpose. For example, OLIGO 4.06 software is useful forthe selection of PCR primer pairs of up to 100 nucleotides each, and forthe analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. Similar primer selection programs have incorporatedadditional features for expanded capabilities. For example, the PrimOUprimer selection program (available to the public from the Genome Centerat University of Texas South West Medical Center, Dallas Tex.) iscapable of choosing specific primers from megabase sequences and is thususeful for designing primers on a genome-wide scope. The Primer3 primerselection program (available to the public from the WhiteheadInstitute/MIT Center for Genome Research, Cambridge Mass.) allows theuser to input a “mispriming library,” in which sequences to avoid asprimer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Centre, Cambridge UK) designsprimers based on multiple sequence alignments, thereby allowingselection of primers that hybridize to either the most conserved orleast conserved regions of aligned nucleic acid sequences. Hence, thisprogram is useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed above.

A “recombinant nucleic acid” is a nucleic acid that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook and Russell (supra). The term recombinant includes nucleicacids that have been altered solely by addition, substitution, ordeletion of a portion of the nucleic acid. Frequently, a recombinantnucleic acid may include a nucleic acid sequence operably linked to apromoter sequence. Such a recombinant nucleic acid may be part of avector that is used, for example, to transform a cell.

Alternatively, such recombinant nucleic acids may be part of a viralvector, e.g., based on a vaccinia virus, that could be use to vaccinatea mammal wherein the recombinant nucleic acid is expressed, inducing aprotective immunological response in the mammal.

A “regulatory element” refers to a nucleic acid sequence usually derivedfrom untranslated regions of a gene and includes enhancers, promoters,introns, and 5′ and 3′ untranslated regions (UTRs). Regulatory elementsinteract with host or viral proteins which control transcription,translation, or RNA stability.

“Reporter molecules” are chemical or biochemical moieties used forlabeling a nucleic acid, amino acid, or antibody. Reporter moleculesinclude radionuclides; enzymes; fluorescent, chemiluminescent, orchromogenic agents; substrates; cofactors; inhibitors; magneticparticles; and other moieties known in the art.

An “RNA equivalent,” in reference to a DNA molecule, is composed of thesame linear sequence of nucleotides as the reference DNA molecule withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining KPP, nucleic acids encoding KPP, or fragments thereof maycomprise a bodily fluid; an extract from a cell, chromosome, organelle;or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, insolution or bound to a substrate; a tissue; a tissue print; etc.

The terms “specific binding” and “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, anantagonist, a small molecule, or any natural or synthetic bindingcomposition. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide comprisingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least about 60% free, preferably atleast about 75% free, and most preferably at least about 90% free fromother components with which they are naturally associated.

A “substitution” refers to the replacement of one or more amino acidresidues or nucleotides by different amino acid residues or nucleotides,respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

A “transcript image” or “expression profile” refers to the collectivepattern of gene expression by a particular cell type or tissue undergiven conditions at a given time.

“Transformation” describes a process by which exogenous DNA isintroduced into a recipient cell. Transformation may occur under naturalor artificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, bacteriophageor viral infection, electroporation, heat shock, lipofection, andparticle bombardment. The term “transformed cells” includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “transgenic organism,” as used herein, is any organism, including butnot limited to animals and plants, in which one or more of the cells ofthe organism contains heterologous nucleic acid introduced by way ofhuman intervention, such as by transgenic techniques well known in theart. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. In another embodiment, the nucleicacid can be introduced by infection with a recombinant viral vector,such as a lentiviral vector (Lois, C. et al. (2002) Science295:868-872). The term genetic manipulation does not include classicalcross-breeding, or in vitro fertilization, but rather is directed to theintroduction of a recombinant DNA molecule. The transgenic organismscontemplated in accordance with the present invention include bacteria,cyanobacteria, fungi, plants and animals. The isolated DNA of thepresent invention can be introduced into the host by methods known inthe art, for example infection, transfection, transformation ortransconjugation. Techniques for transferring the DNA of the presentinvention into such organisms are widely known and provided inreferences such as Sambrook and Russell (supra).

A “variant” of a particular nucleic acid sequence is defined as anucleic acid sequence having at least 40% sequence identity to theparticular nucleic acid sequence over a certain length of one of thenucleic acid sequences using blastn with the “BLAST 2 Sequences” toolVersion 2.0.9 (May-07-1999) set at default parameters. Such a pair ofnucleic acids may show, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% or greater sequence identityover a certain defined length. A variant may be described as, forexample, an “allelic” (as defined above), “splice,” “species,” or“polymorphic” variant. A splice variant may have significant identity toa reference molecule, but will generally have a greater or lesser numberof polynucleotides due to alternate splicing during mRNA processing. Thecorresponding polypeptide may possess additional functional domains orlack domains that are present in the reference molecule. Speciesvariants are polynucleotides that vary from one species to another. Theresulting polypeptides will generally have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) in which the polynucleotide sequencevaries by one nucleotide base. The presence of SNPs may be indicativeof, for example, a certain population, a disease state, or a propensityfor a disease state.

A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity or sequencesimilarity to the particular polypeptide sequence over a certain lengthof one of the polypeptide sequences using blastp with the “BLAST 2Sequences” tool Version 2.0.9 (May-07-1999) set at default parameters.Such a pair of polypeptides may show, for example, at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% or greatersequence identity or sequence similarity over a certain defined lengthof one of the polypeptides.

THE INVENTION

Various embodiments of the invention include new human kinases andphosphatases (KPP), the polynucleotides encoding KPP, and the use ofthese compositions for the diagnosis, treatment, or prevention ofcardiovascular diseases, immune system disorders, neurologicaldisorders, disorders affecting growth and development, lipid disorders,cell proliferative disorders, and cancers.

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide embodiments of the invention. Each polynucleotide andits corresponding polypeptide are correlated to a single Incyte projectidentification number (Incyte Project ID). Each polypeptide sequence isdenoted by both a polypeptide sequence identification number(Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number(Incyte Polypeptide ID) as shown. Each polynucleotide sequence isdenoted by both a polynucleotide sequence identification number(Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensussequence number (Incyte Polynucleotide ID) as shown. Column 6 shows theIncyte ID numbers of physical, full length clones corresponding to thepolypeptide and polynucleotide sequences of the invention. The fulllength clones encode polypeptides which have at least 95% sequenceidentity to the polypeptide sequences shown in column 3.

Table 2 shows sequences with homology to polypeptide embodiments of theinvention as identified by BLAST analysis against the GenBank protein(genpept) database and the PROTEOME database. Columns 1 and 2 show thepolypeptide sequence identification number (Polypeptide SEQ ID NO:) andthe corresponding Incyte polypeptide sequence number (Incyte PolypeptideID) for polypeptides of the invention. Column 3 shows the GenBankidentification number (GenBank ID NO:) of the nearest GenBank homologand the PROTEOME database identification numbers (PROTEOME ID NO:) ofthe nearest PROTEOME database homologs. Column 4 shows the probabilityscores for the matches between each polypeptide and its homolog(s).Column 5 shows the annotation of the GenBank and PROTEOME databasehomolog(s) along with relevant citations where applicable, all of whichare expressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of theinvention. Columns 1 and 2 show the polypeptide sequence identificationnumber (SEQ ID NO:) and the corresponding Incyte polypeptide sequencenumber (Incyte Polypeptide ID) for each polypeptide of the invention.Column 3 shows the number of amino acid residues in each polypeptide.Column 4 shows amino acid residues comprising signature sequences,domains, motifs, potential phosphorylation sites, and potentialglycosylation sites. Column 5 shows analytical methods for proteinstructure/function analysis and in some cases, searchable databases towhich the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of theinvention, and these properties establish that the claimed polypeptidesare kinases and phosphatases. For example, SEQ ID NO:11 is 78%identical, from residue M1 to residue W1219, to mouse NIK (GenBank IDg1872546) as determined by the Basic Local Alignment Search Tool(BLAST). (See Table 2.) The BLAST probability score is 0.0, whichindicates the probability of obtaining the observed polypeptide sequencealignment by chance. SEQ ID NO:11 also has homology to proteins thatactivate the c-Jun N-terminal kinase (Mapk8) signaling pathway, and aremitogen-activated protein kinase kinase kinase kinases (MAP4K), asdetermined by BLAST analysis using the PROTEOME database. SEQ ID NO:11also contains a CNH domain, a protein kinase domain, a domain found inNIK1-like kinases, and a serine/threonine kinase catalytic domain, asdetermined by searching for statistically significant matches in thehidden Markov model (HMM)-based PFAM and SMART databases of conservedprotein families/domains. (See Table 3.) Data from BLIMPS, MOTIFS, andPROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMOdatabases, provide further corroborative evidence that SEQ ID NO:11 is aprotein kinase.

As another example, SEQ ID NO:15 is 99% identical, from residue E124 toresidue I750, to human lymphoid phosphatase LyP1 (GenBank ID g4100632)as determined by the Basic Local Alignment Search Tool (BLAST). (SeeTable 2.) The BLAST probability score is 0.0, which indicates theprobability of obtaining the observed polypeptide sequence alignment bychance. SEQ ID NO:15 also has homology to proteins that may be involvedin T-cell development and are required for B-cell antigenreceptor-mediated growth arrest and apoptosis and are protein tyrosinephosphatase non-receptors, as determined by BLAST analysis using thePROTEOME database. SEQ ID NO:15 also contains a protein-tyrosinephosphatase domain, a protein-tyrosine phosphatase catalytic domain, anda protein-tyrosine phosphatase catalytic motif domain as determined bysearching for statistically significant matches in the hidden Markovmodel (HMM)-based SMART and PFAM databases of conserved proteinfamilies/domains. (See Table 3.) Data from BLIMPS, MOTIFS, andPROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMOdatabases, provide further corroborative evidence that SEQ ID NO:15 is aprotein-tyrosine phosphatase.

As another example, SEQ ID NO:24 is 99% identical, from residue M1 toresidue K487, to human apyrase (GenBank ID g4583675) as determined bythe Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 3.7e-264, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. SEQ IDNO:24 also has homology to proteins that are localized to thelysosomal/autophagic vacuoles and are apyrase proteins, as determined byBLAST analysis using the PROTEOME database. SEQ ID NO:24 also contains aGDA1/CD39 (nucleoside phosphatase family) domain as determined bysearching for statistically significant matches in the hidden Markovmodel (HMM)-based PFAM database of conserved protein families/domains.(See Table 3.) Data from BLIMPS and BLAST analyses against the PRODOMand DOMO databases, provide further corroborative evidence that SEQ IDNO:24 is a nucleoside phosphatase.

As another example, SEQ ID NO:27 is 97% identical, from residue M1 toresidue G76, to human SKRP1 (GenBank ID g18148911) as determined by theBasic Local Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 5.7e-35, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. SEQ IDNO:27 also has homology to proteins that dephosphorylate phosphotyrosineand phosphoserine, inactivate MAPK, and are proteins containing two dualspecificity phosphatase catalytic domains, as determined by BLASTanalysis using the PROTEOME database. Data from BLIMPS analyses providefurther corroborative evidence that SEQ ID NO:27 is a dual specificityphosphatase.

As another example, SEQ ID NO:28 is 98% identical, from residue M1 toresidue S449, to human protein phosphatase 4 regulatory subunit 2(GenBank ID g8250239) as determined by the Basic Local Alignment SearchTool (BLAST). (See Table 2.) The BLAST probability score is 1.4E-241,which indicates the probability of obtaining the observed polypeptidesequence alignment by chance. SEQ ID NO:28 also has homology to humanprotein phosphatase 4 regulatory subunit 2, as determined by BLASTanalysis using the PROTEOME database. The foregoing provide evidencethat SEQ ID NO:28 is a protein phosphatase regulatory subunit.

As another example, SEQ ID NO:34 is 93% identical, from residue E39 toresidue 1490, to human multifunctional calcium/calmodulin-dependentprotein kinase II delta2 isoform (GenBank ID g4426595) as determined bythe Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 9.0e-255, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. SEQ IDNO:34 also has homology to calcium-calmodulin dependent protein kinaseII delta, a member of the multifunctional CAMKII family involved in Ca2+regulated processes, of which the alternative form delta 3 isspecifically upregulated in the myocardium of patients with heartfailure, as determined by BLAST analysis using the PROTEOME database.SEQ ID NO:34 also contains a protein kinase domain and aserine/threonine protein kinase catalytic domain as determined bysearching for statistically significant matches in the hidden Markovmodel (HMM)-based PFAM and SMART databases of conserved proteinfamilies/domains. (See Table 3.) Data from BLIMPS, MOTIFS, andPROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMOdatabases, provide further corroborative evidence that SEQ ID NO:34 is acalcium-calmodulin dependent protein kinase. The foregoing providesevidence that SEQ ID NO:34 is a calcium-calmodulin dependent proteinkinase.

SEQ ID NO:1-10, SEQ ID NO:12-14, SEQ ID NO:16-23, SEQ ID NO:25-26, SEQID NO:29-33, and SEQ ID NO:35-43 were analyzed and annotated in asimilar manner. The algorithms and parameters for the analysis of SEQ IDNO:1-43 are described in Table 7.

As shown in Table 4, the full length polynucleotide embodiments wereassembled using cDNA sequences or coding (exon) sequences derived fromgenomic DNA, or any combination of these two types of sequences. Column1 lists the polynucleotide sequence identification number(Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotideconsensus sequence number (Incyte ID) for each polynucleotide of theinvention, and the length of each polynucleotide sequence in basepairs.Column 2 shows the nucleotide start (5′) and stop (3′) positions of thecDNA and/or genomic sequences used to assemble the full lengthpolynucleotide embodiments, and of fragments of the polynucleotideswhich are useful, for example, in hybridization or amplificationtechnologies that identify SEQ ID NO:44-86 or that distinguish betweenSEQ ID NO:44-86 and related polynucleotides.

The polynucleotide fragments described in Column 2 of Table 4 may referspecifically, for example, to Incyte cDNAs derived from tissue-specificcDNA libraries or from pooled cDNA libraries. Alternatively, thepolynucleotide fragments described in column 2 may refer to GenBankcDNAs or ESTs which contributed to the assembly of the full lengthpolynucleotides. In addition, the polynucleotide fragments described incolumn 2 may identify sequences derived from the ENSEMBL (The SangerCentre, Cambridge, UK) database (i.e., those sequences including thedesignation “ENST”). Alternatively, the polynucleotide fragmentsdescribed in column 2 may be derived from the NCBI RefSeq NucleotideSequence Records Database (i.e., those sequences including thedesignation “NM” or “NT”) or the NCBI RefSeq Protein Sequence Records(i.e., those sequences including the designation “NP”). Alternatively,the polynucleotide fragments described in column 2 may refer toassemblages of both cDNA and Genscan-predicted exons brought together byan “exon stitching” algorithm. For example, a polynucleotide sequenceidentified as FL_XXXXXX_N₁ _(—) N₂ _(—) YYYYY_N₃ _(—) N₄ represents a“stitched” sequence in which XXXXXX is the identification number of thecluster of sequences to which the algorithm was applied, and YYYYY isthe number of the prediction generated by the algorithm, andN_(1,2,3 . . .) , if present, represent specific exons that may havebeen manually edited during analysis (See Example V). Alternatively, thepolynucleotide fragments in column 2 may refer to assemblages of exonsbrought together by an “exon-stretching” algorithm. For example, apolynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_(—)1_N is a“stretched” sequence, with XXXXXX being the Incyte projectidentification number, gAAAAA being the GenBank identification number ofthe human genomic sequence to which the “exon-stretching” algorithm wasapplied, gBBBBB being the GenBank identification number or NCBI RefSeqidentification number of the nearest GenBank protein homolog, and Nreferring to specific exons (See Example V). In instances where a RefSeqsequence was used as a protein homolog for the “exon-stretching”algorithm, a RefSeq identifier (denoted by “NM,” “NP,” or “NT”) may beused in place of the GenBank identifier (i.e., gBBBBB).

Alternatively, a prefix identifies component sequences that werehand-edited, predicted from genomic DNA sequences, or derived from acombination of sequence analysis methods. The following Table listsexamples of component sequence prefixes and corresponding sequenceanalysis methods associated with the prefixes (see Example IV andExample V).

Prefix Type of analysis and/or examples of programs GNN, GFG, Exonprediction from genomic sequences using, for ENST example, GENSCAN(Stanford University, CA, USA) or FGENES (Computer Genomics Group, TheSanger Centre, Cambridge, UK). GBI Hand-edited analysis of genomicsequences. FL Stitched or stretched genomic sequences (see Example V).INCY Full length transcript and exon prediction from mapping of ESTsequences to the genome. Genomic location and EST composition data arecombined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverageshown in Table 4 was obtained to confirm the final consensuspolynucleotide sequence, but the relevant Incyte cDNA identificationnumbers are not shown.

Table 5 shows the representative cDNA libraries for those full lengthpolynucleotides which were assembled using Incyte cDNA sequences. Therepresentative cDNA library is the Incyte cDNA library which is mostfrequently represented by the Incyte cDNA sequences which were used toassemble and confirm the above polynucleotides. The tissues and vectorswhich were used to construct the cDNA libraries shown in Table 5 aredescribed in Table 6.

Table 8 shows single nucleotide polymorphisms (SNPs) found inpolynucleotide sequences of the invention, along with allele frequenciesin different human populations. Columns 1 and 2 show the polynucleotidesequence identification number (SEQ ID NO:) and the corresponding Incyteproject identification number (ND) for polynucleotides of the invention.Column 3 shows the Incyte identification number for the EST in which theSNP was detected (EST ID), and column 4 shows the identification numberfor the SNP (SNP ID). Column 5 shows the position within the ESTsequence at which the SNP is located (EST SNP), and column 6 shows theposition of the SNP within the full-length polynucleotide sequence (CB 1SNP). Column 7 shows the allele found in the EST sequence. Columns 8 and9 show the two alleles found at the SNP site. Column 10 shows the aminoacid encoded by the codon including the SNP site, based upon the allelefound in the EST. Columns 11-14 show the frequency of allele 1 in fourdifferent human populations. An entry of n/d (not detected) indicatesthat the frequency of allele 1 in the population was too low to bedetected, while n/a (not available) indicates that the allele frequencywas not determined for the population.

The invention also encompasses KPP variants. Various embodiments of KPPvariants can have at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% amino acid sequence identity to the KPP amino acidsequence, and can contain at least one functional or structuralcharacteristic of KPP.

Various embodiments also encompass polynucleotides which encode KPP. Ina particular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO:44-86, which encodes KPP. The polynucleotide sequences of SEQ IDNO:44-86, as presented in the Sequence Listing, embrace the equivalentRNA sequences, wherein occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The invention also encompasses variants of a polynucleotide encodingKPP. In particular, such a variant polynucleotide will have at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%polynucleotide sequence identity to a polynucleotide encoding KPP. Aparticular aspect of the invention encompasses a variant of apolynucleotide comprising a sequence selected from the group consistingof SEQ ID NO:44-86 which has at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% polynucleotide sequence identity to a nucleicacid sequence selected from the group consisting of SEQ ID NO:44-86. Anyone of the polynucleotide variants described above can encode apolypeptide which contains at least one functional or structuralcharacteristic of KPP.

In addition, or in the alternative, a polynucleotide variant of theinvention is a splice variant of a polynucleotide encoding KPP. A splicevariant may have portions which have significant sequence identity to apolynucleotide encoding KPP, but will generally have a greater or lessernumber of nucleotides due to additions or deletions of blocks ofsequence arising from alternate splicing during mRNA processing. Asplice variant may have less than about 70%, or alternatively less thanabout 60%, or alternatively less than about 50% polynucleotide sequenceidentity to a polynucleotide encoding KPP over its entire length;however, portions of the splice variant will have at least about 70%, oralternatively at least about 85%, or alternatively at least about 95%,or alternatively 100% polynucleotide sequence identity to portions ofthe polynucleotide encoding KPP. For example, a polynucleotidecomprising a sequence of SEQ ID NO:48, a polynucleotide comprising asequence of SEQ ID NO:49 and a polynucleotide comprising a sequence ofSEQ ID NO:50 are splice variants of each other; a polynucleotidecomprising a sequence of SEQ ID NO:75 and a polynucleotide comprising asequence of SEQ ID NO:76 are splice variants of each other; apolynucleotide comprising a sequence of SEQ ID NO:77 and apolynucleotide comprising a sequence of SEQ ID NO:78 are splice variantsof each other; a polynucleotide comprising a sequence of SEQ ID NO:79and a polynucleotide comprising a sequence of SEQ ID NO:80 are splicevariants of each other; a polynucleotide comprising a sequence of SEQ IDNO:57 and a polynucleotide comprising a sequence of SEQ ID NO:62 aresplice variants of each other, and a polynucleotide comprising asequence of SEQ ID NO:68 and a polynucleotide comprising a sequence ofSEQ ID NO:69 are splice variants of each other. Any one of the splicevariants described above can encode a polypeptide which contains atleast one functional or structural characteristic of KPP.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding KPP, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring KPP, and all suchvariations are to be considered as being specifically disclosed.

Although polynucleotides which encode KPP and its variants are generallycapable of hybridizing to polynucleotides encoding naturally occurringKPP under appropriately selected conditions of stringency, it may beadvantageous to produce polynucleotides encoding KPP or its derivativespossessing a substantially different codon usage, e.g., inclusion ofnon-naturally occurring codons. Codons may be selected to increase therate at which expression of the peptide occurs in a particularprokaryotic or eukaryotic host in accordance with the frequency withwhich particular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence encoding KPP and itsderivatives without altering the encoded amino acid sequences includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

The invention also encompasses production of polynucleotides whichencode KPP and KPP derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic polynucleotide maybe inserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a polynucleotideencoding KPP or any fragment thereof.

Embodiments of the invention can also include polynucleotides that arecapable of hybridizing to the claimed polynucleotides, and, inparticular, to those having the sequences shown in SEQ ID NO:44-86 andfragments thereof, under various conditions of stringency (Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R.(1987) Methods Enzymol. 152:507-511). Hybridization conditions,including annealing and wash conditions, are described in “Definitions.”

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (USBiochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems),thermostable T7 polymerase (Amersham Biosciences, Piscataway N.J.), orcombinations of polymerases and proofreading exonucleases such as thosefound in the ELONGASE amplification system (Invitrogen, CarlsbadCalif.). Preferably, sequence preparation is automated with machinessuch as the MICROLAB 2200 liquid transfer system (Hamilton, Reno Nev.),PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABI CATALYST800 thermal cycler (Applied Biosystems). Sequencing is then carried outusing either the ABI 373 or 377 DNA sequencing system (AppliedBiosystems), the MEGABACE 1000 DNA sequencing system (AmershamBiosciences), or other systems known in the art. The resulting sequencesare analyzed using a variety of algorithms which are well known in theart (Ausubel et al., supra, ch. 7; Meyers, R. A. (1995) MolecularBiology and Biotechnology, Wiley V C H, New York N.Y., pp. 856-853).

The nucleic acids encoding KPP may be extended utilizing a partialnucleotide sequence and employing various PCR-based methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector (Sarkar, G.(1993) PCR Methods Applic. 2:318-322). Another method, inverse PCR, usesprimers that extend in divergent directions to amplify unknown sequencefrom a circularized template. The template is derived from restrictionfragments comprising a known genomic locus and surrounding sequences(Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). A third method,capture PCR, involves PCR amplification of DNA fragments adjacent toknown sequences in human and yeast artificial chromosome DNA(Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). In thismethod, multiple restriction enzyme digestions and ligations may be usedto insert an engineered double-stranded sequence into a region ofunknown sequence before performing PCR. Other methods which may be usedto retrieve unknown sequences are known in the art (Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (BD Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppliedBiosystems), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotides or fragmentsthereof which encode KPP may be cloned in recombinant DNA molecules thatdirect expression of KPP, or fragments or functional equivalentsthereof, in appropriate host cells. Due to the inherent degeneracy ofthe genetic code, other polynucleotides which encode substantially thesame or a functionally equivalent polypeptides may be produced and usedto express KPP.

The polynucleotides of the invention can be engineered, using methodsgenerally known in the art in order to alter KPP-encoding sequences fora variety of purposes including, but not limited to, modification of thecloning, processing, and/or expression of the gene product. DNAshuffling by random fragmentation and PCR reassembly of gene fragmentsand synthetic oligonucleotides may be used to engineer the nucleotidesequences. For example, oligonucleotide-mediated site-directedmutagenesis may be used to introduce mutations that create newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

The nucleotides of the present invention may be subjected to DNAshuffling techniques such as MOLECULARBREEDING (Maxygen Inc., SantaClara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al.(1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat.Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.14:315-319) to alter or improve the biological properties of KPP, suchas its biological or enzymatic activity or its ability to bind to othermolecules or compounds. DNA shuffling is a process by which a library ofgene variants is produced using PCR-mediated recombination of genefragments. The library is then subjected to selection or screeningprocedures that identify those gene variants with the desiredproperties. These preferred variants may then be pooled and furthersubjected to recursive rounds of DNA shuffling and selection/screening.Thus, genetic diversity is created through “artificial” breeding andrapid molecular evolution. For example, fragments of a single genecontaining random point mutations may be recombined, screened, and thenreshuffled until the desired properties are optimized. Alternatively,fragments of a given gene may be recombined with fragments of homologousgenes in the same gene family, either from the same or differentspecies, thereby maximizing the genetic diversity of multiple naturallyoccurring genes in a directed and controllable manner.

In another embodiment, polynucleotides encoding KPP may be synthesized,in whole or in part, using one or more chemical methods well known inthe art (Caruthers, M. H. et al. (1980) Nucleic Acids Symp. Ser.7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).Alternatively, KPP itself or a fragment thereof may be synthesized usingchemical methods known in the art. For example, peptide synthesis can beperformed using various solution-phase or solid-phase techniques(Creighton, T. (1984) Proteins, Structures and Molecular Properties, WHFreeman, New York N.Y., pp. 55-60; Roberge, J. Y. et al. (1995) Science269:202-204). Automated synthesis may be achieved using the ABI 431Apeptide synthesizer (Applied Biosystems). Additionally, the amino acidsequence of KPP, or any part thereof, may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant polypeptide or a polypeptide having asequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography (Chiez, R. M. and F. Z. Regnier (1990)Methods Enzymol. 182:392-421). The composition of the synthetic peptidesmay be confirmed by amino acid analysis or by sequencing (Creighton,supra, pp. 28-53).

In order to express a biologically active KPP, the polynucleotidesencoding KPP or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5′ and 3′ untranslated regions in the vector and in polynucleotidesencoding KPP. Such elements may vary in their strength and specificity.Specific initiation signals may also be used to achieve more efficienttranslation of polynucleotides encoding KPP. Such signals include theATG initiation codon and adjacent sequences, e.g. the Kozak sequence. Incases where a polynucleotide sequence encoding KPP and its initiationcodon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used (Scharf,D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing polynucleotides encoding KPP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination (Sambrook and Russell,supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15).

A variety of expression vector/host systems may be utilized to containand express polynucleotides encoding KPP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrookand Russell, supra; Ausubel et al., supra; Van Heeke, G. and S. M.Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; TheMcGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, NewYork N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad.Sci. USA 81:3655-3659; Harrington, J. J. et al. (1997) Nat. Genet.15:345-355). Expression vectors derived from retroviruses, adenoviruses,or herpes or vaccinia viruses, or from various bacterial plasmids, maybe used for delivery of polynucleotides to the targeted organ, tissue,or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther.5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344;Buller, R. M. et al. (1985) Nature 317:813-815; McGregor, D. P. et al.(1994) Mol. Immunol. 31:219-226; Verma, I. M. and N. Sonia (1997) Nature389:239-242). The invention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotides encodingKPP. For example, routine cloning, subcloning, and propagation ofpolynucleotides encoding KPP can be achieved using a multifunctional E.coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1plasmid (Invitrogen). Ligation of polynucleotides encoding KPP into thevector's multiple cloning site disrupts the lacZ gene, allowing acolorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.264:5503-5509). When large quantities of KPP are needed, e.g. for theproduction of antibodies, vectors which direct high level expression ofKPP may be used. For example, vectors containing the strong, inducibleSP6 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of KPP. A number ofvectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH promoters, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign polynucleotide sequences intothe host genome for stable propagation (Ausubel et al, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, C. A. et al.(1994) Bio/Technology 12:181-184).

Plant systems may also be used for expression of KPP. Transcription ofpolynucleotides encoding KPP may be driven by viral promoters, e.g., the35S and 19S promoters of CaMV used alone or in combination with theomega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; Winter, J.et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructscan be introduced into plant cells by direct DNA transformation orpathogen-mediated transfection (The McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York N.Y., pp. 191-196).

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,polynucleotides encoding KPP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses KPP in host cells (Logan, J. and T. Shenk (1984) Proc. Natl.Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, suchas the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells. SV40 or EBV-based vectors may alsobe used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes (Harrington, J. J. et al. (1997)Nat. Genet. 15:345-355).

For long term production of recombinant proteins in mammalian systems,stable expression of KPP in cell lines is preferred. For example,polynucleotides encoding KPP can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk′ and apr′ cells, respectively (Wigler, M. et al. (1977) Cell11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG-418; and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Wigler, M. et al.(1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. etal. (1981) J. Mol. Biol. 150:1-14). Additional selectable genes havebeen described, e.g., tipB and hisD, which alter cellular requirementsfor metabolites (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.Acad. Sci. USA 85:8047-8051). Visible markers, e.g., anthocyanins, greenfluorescent proteins (GFP; BD Clontech), β-glucuronidase and itssubstrate β-glucuronide, or luciferase and its substrate luciferin maybe used. These markers can be used not only to identify transformants,but also to quantify the amount of transient or stable proteinexpression attributable to a specific vector system (Rhodes, C.A. (1995)Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encoding KPPis inserted within a marker gene sequence, transformed cells containingpolynucleotides encoding KPP can be identified by the absence of markergene function. Alternatively, a marker gene can be placed in tandem witha sequence encoding KPP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

In general, host cells that contain the polynucleotide encoding KPP andthat express KPP may be identified by a variety of procedures known tothose of skill in the art. These procedures include, but are not limitedto, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of KPPusing either specific polyclonal or monoclonal antibodies are known inthe art. Examples of such techniques include enzyme-linked immunosorbentassays (ELISAs), radioimmunoassays (RIM), and fluorescence activatedcell sorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on KPP ispreferred, but a competitive binding assay may be employed. These andother assays are well known in the art (Hampton, R. et al. (1990)Serological Methods, a Laboratory Manual, APS Press, St. Paul Minn.,Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology,Greene Pub. Associates and Wiley-Interscience, New York N.Y.; Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding KPP includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, polynucleotides encoding KPP,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by AmershamBiosciences, Promega (Madison Wis.), and US Biochemical. Suitablereporter molecules or labels which may be used for ease of detectioninclude radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with polynucleotides encoding KPP may be culturedunder conditions suitable for the expression and recovery of the proteinfrom cell culture. The protein produced by a transformed cell may besecreted or retained intracellularly depending on the sequence and/orthe vector used. As will be understood by those of skill in the art,expression vectors containing polynucleotides which encode KPP may bedesigned to contain signal sequences which direct secretion of KPPthrough a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted polynucleotides or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” or “pro” form ofthe protein may also be used to specify protein targeting, folding,and/or activity. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and W138) are available fromthe American Type Culture Collection (ATCC, Manassas Va.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

In another embodiment of the invention, natural, modified, orrecombinant polynucleotides encoding KPP may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric KPPprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of KPP activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His (SEQ ID NO: 87), FLAG, c-myc, and hemagglutinin (HA). GST, MBP,Trx, CBP, and 6-His (SEQ ID NO: 87) enable purification of their cognatefusion proteins on immobilized glutathione, maltose, phenylarsine oxide,calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, andhemagglutinin (HA) enable immunoaffinity purification of fusion proteinsusing commercially available monoclonal and polyclonal antibodies thatspecifically recognize these epitope tags. A fusion protein may also beengineered to contain a proteolytic cleavage site located between theKPP encoding sequence and the heterologous protein sequence, so that KPPmay be cleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel et al. (supra, ch. 10 and 16). A variety of commerciallyavailable kits may also be used to facilitate expression andpurification of fusion proteins.

In another embodiment, synthesis of radiolabeled KPP may be achieved invitro using the TNT rabbit reticulocyte lysate or wheat germ extractsystem (Promega). These systems couple transcription and translation ofprotein-coding sequences operably associated with the T7, T3, or SP6promoters. Translation takes place in the presence of a radiolabeledamino acid precursor, for example, ³⁵S-methionine.

KPP, fragments of KPP, or variants of KPP may be used to screen forcompounds that specifically bind to KPP. One or more test compounds maybe screened for specific binding to KPP. In various embodiments, 1, 2,3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened forspecific binding to KPP. Examples of test compounds can includeantibodies, anticalins, oligonucleotides, proteins (e.g., ligands orreceptors), or small molecules.

In related embodiments, variants of KPP can be used to screen forbinding of test compounds, such as antibodies, to KPP, a variant of KPP,or a combination of KPP and/or one or more variants KPP. In anembodiment, a variant of KPP can be used to screen for compounds thatbind to a variant of KPP, but not to KPP having the exact sequence of asequence of SEQ ID NO:1-43. KPP variants used to perform such screeningcan have a range of about 50% to about 99% sequence identity to KPP,with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95%sequence identity.

In an embodiment, a compound identified in a screen for specific bindingto KPP can be closely related to the natural ligand of KPP, e.g., aligand or fragment thereof, a natural substrate, a structural orfunctional mimetic, or a natural binding partner (Coligan, J. E. et al.(1991) Current Protocols in Immunology 1(2):Chapter 5). In anotherembodiment, the compound thus identified can be a natural ligand of areceptor KPP (Howard, A. D. et al. (2001) Trends Pharmacol. Sci.22:132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).

In other embodiments, a compound identified in a screen for specificbinding to KPP can be closely related to the natural receptor to whichKPP binds, at least a fragment of the receptor, or a fragment of thereceptor including all or a portion of the ligand binding site orbinding pocket. For example, the compound may be a receptor for KPPwhich is capable of propagating a signal, or a decoy receptor for KPPwhich is not capable of propagating a signal (Ashkenazi, A. and V. M.Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al.(2001) Trends Immunol. 22:328-336). The compound can be rationallydesigned using known techniques. Examples of such techniques includethose used to construct the compound etanercept (ENBREL; Amgen Inc.,Thousand Oaks Calif.), which is efficacious for treating rheumatoidarthritis in humans. Etanercept is an engineered p75 tumor necrosisfactor (TNF) receptor dimer linked to the Fc portion of human IgG₁(Taylor, P. C. et al. (2001) Curr. Opin. Immunol 13:611-616).

In one embodiment, two or more antibodies having similar or,alternatively, different specificities can be screened for specificbinding to KPP, fragments of KPP, or variants of KPP. The bindingspecificity of the antibodies thus screened can thereby be selected toidentify particular fragments or variants of KPP. In one embodiment, anantibody can be selected such that its binding specificity allows forpreferential identification of specific fragments or variants of KPP. Inanother embodiment, an antibody can be selected such that its bindingspecificity allows for preferential diagnosis of a specific disease orcondition having increased, decreased, or otherwise abnormal productionof KPP.

In an embodiment, anticalins can be screened for specific binding, toKPP, fragments of KPP, or variants of KPP. Anticalins are ligand-bindingproteins that have been constructed based on a lipocalin scaffold(Weiss, G. A. and H. B. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra,A. (2001) J. Biotechnol. 74:257-275). The protein architecture oflipocalins can include a beta-barrel having eight antiparallelbeta-strands, which supports four loops at its open end. These loopsform the natural ligand-binding site of the lipocalins, a site which canbe re-engineered in vitro by amino acid substitutions to impart novelbinding specificities. The amino acid substitutions can be made usingmethods known in the art or described herein, and can includeconservative substitutions (e.g., substitutions that do not alterbinding specificity) or substitutions that modestly, moderately, orsignificantly alter binding specificity.

In one embodiment, screening for compounds which specifically bind to,stimulate, or inhibit KPP involves producing appropriate cells whichexpress KPP, either as a secreted protein or on the cell membrane.Preferred cells can include cells from mammals, yeast, Drosophila, or E.coli. Cells expressing KPP or cell membrane fractions which contain KPPare then contacted with a test compound and binding, stimulation, orinhibition of activity of either KPP or the compound is analyzed.

An assay may simply test binding of a test compound to the polypeptide,wherein binding is detected by a fluorophore, radioisotope, enzymeconjugate, or other detectable label. For example, the assay maycomprise the steps of combining at least one test compound with KPP,either in solution or affixed to a solid support, and detecting thebinding of KPP to the compound. Alternatively, the assay may detect ormeasure binding of a test compound in the presence of a labeledcompetitor. Additionally, the assay may be carried out using cell-freepreparations, chemical libraries, or natural product mixtures, and thetest compound(s) may be free in solution or affixed to a solid support.

An assay can be used to assess the ability of a compound to bind to itsnatural ligand and/or to inhibit the binding of its natural ligand toits natural receptors. Examples of such assays include radio-labelingassays such as those described in U.S. Pat. No. 5,914,236 and U.S. Pat.No. 6,372,724. In a related embodiment, one or more amino acidsubstitutions can be introduced into a polypeptide compound (such as areceptor) to improve or alter its ability to bind to its natural ligands(Matthews, D. J. and J. A. Wells. (1994) Chem. Biol. 1:25-30). Inanother related embodiment, one or more amino acid substitutions can beintroduced into a polypeptide compound (such as a ligand) to improve oralter its ability to bind to its natural receptors (Cunningham, B. C.and J. A. Wells (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman,H. B. et al. (1991) J. Biol. Chem. 266:10982-10988).

KPP, fragments of KPP, or variants of KPP may be used to screen forcompounds that modulate the activity of KPP. Such compounds may includeagonists, antagonists, or partial or inverse agonists. In oneembodiment, an assay is performed under conditions permissive for KPPactivity, wherein KPP is combined with at least one test compound, andthe activity of KPP in the presence of a test compound is compared withthe activity of KPP in the absence of the test compound. A change in theactivity of KPP in the presence of the test compound is indicative of acompound that modulates the activity of KPP. Alternatively, a testcompound is combined with an in vitro or cell-free system comprising KPPunder conditions suitable for KPP activity, and the assay is performed.In either of these assays, a test compound which modulates the activityof KPP may do so indirectly and need not come in direct contact with thetest compound. At least one and up to a plurality of test compounds maybe screened.

In another embodiment, polynucleotides encoding KPP or their mammalianhomologs may be “knocked out” in an animal model system using homologousrecombination in embryonic stem (ES) cells. Such techniques are wellknown in the art and are useful for the generation of animal models ofhuman disease (see, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No.5,767,337). For example, mouse ES cells, such as the mouse 129/SvJ cellline, are derived from the early mouse embryo and grown in culture. TheES cells are transformed with a vector containing the gene of interestdisrupted by a marker gene, e.g., the neomycin phosphotransferase gene(neo; Capecchi, M. R. (1989) Science 244:1288-1292). The vectorintegrates into the corresponding region of the host genome byhomologous recombination. Alternatively, homologous recombination takesplace using the Cre-1oxP system to knockout a gene of interest in atissue- or developmental stage-specific manner (Marth, J. D. (1996)Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997) Nucleic AcidsRes. 25:4323-4330). Transformed ES cells are identified andmicroinjected into mouse cell blastocysts such as those from the C57BL/6mouse strain. The blastocysts are surgically transferred topseudopregnant dams, and the resulting chimeric progeny are genotypedand bred to produce heterozygous or homozygous strains. Transgenicanimals thus generated may be tested with potential therapeutic or toxicagents.

Polynucleotides encoding KPP may also be manipulated in vitro in EScells derived from human blastocysts. Human ES cells have the potentialto differentiate into at least eight separate cell lineages includingendoderm, mesoderm, and ectodermal cell types. These cell lineagesdifferentiate into, for example, neural cells, hematopoietic lineages,and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding KPP can also be used to create “knockin”humanized animals (pigs) or transgenic animals (mice or rats) to modelhuman disease. With knockin technology, a region of a polynucleotideencoding KPP is injected into animal ES cells, and the injected sequenceintegrates into the animal cell genome. Transformed cells are injectedinto blastulae, and the blastulae are implanted as described above.Transgenic progeny or inbred lines are studied and treated withpotential pharmaceutical agents to obtain information on treatment of ahuman disease. Alternatively, a mammal inbred to overexpress KPP, e.g.,by secreting KPP in its milk, may also serve as a convenient source ofthat protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).

Therapeutics

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of KPP and kinases and phosphatases.In addition, examples of tissues expressing KPP can be found in Table 6and can also be found in Example XI. Therefore, KPP appears to play arole in cardiovascular diseases, immune system disorders, neurologicaldisorders, disorders affecting growth and development, lipid disorders,cell proliferative disorders, and cancers. In the treatment of disordersassociated with increased KPP expression or activity, it is desirable todecrease the expression or activity of KPP. In the treatment ofdisorders associated with decreased KPP expression or activity, it isdesirable to increase the expression or activity of KPP.

Therefore, in one embodiment, KPP or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of KPP.

Examples of such disorders include, but are not limited to, acardiovascular disease such as arteriovenous fistula, atherosclerosis,hypertension, vasculitis, Raynaud's disease, aneurysms, arterialdissections, varicose veins, thrombophlebitis and phlebothrombosis,vascular tumors, and complications of thrombolysis, balloon angioplasty,vascular replacement, and coronary artery bypass graft surgery,congestive heart failure, ischemic heart disease, angina pectoris,myocardial infarction, hypertensive heart disease, degenerative valvularheart disease, calcific aortic valve stenosis, congenitally bicuspidaortic valve, mitral annular calcification, mitral valve prolapse,rheumatic fever and rheumatic heart disease, infective endocarditis,nonbacterial thrombotic endocarditis, endocarditis of systemic lupuserythematosus, carcinoid heart disease, cardiomyopathy, myocarditis,pericarditis, neoplastic heart disease, congenital heart disease, andcomplications of cardiac transplantation, congenital lung anomalies,atelectasis, pulmonary congestion and edema, pulmonary embolism,pulmonary hemorrhage, pulmonary infarction, pulmonary hypertension,vascular sclerosis, obstructive pulmonary disease, restrictive pulmonarydisease, chronic obstructive pulmonary disease, emphysema, chronicbronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viraland mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuseinterstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonaryfibrosis, desquamative interstitial pneumonitis, hypersensitivitypneumonitis, pulmonary eosinophilia bronchiolitis obliterans-organizingpneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture'ssyndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement incollagen-vascular disorders, pulmonary alveolar proteinosis, lungtumors, inflammatory and noninflammatory pleural effusions,pneumothorax, pleural tumors, drug-induced lung disease,radiation-induced lung disease, and complications of lungtransplantation; an immune system disorder such as acquiredimmunodeficiency syndrome (AIDS), Addison's disease, adult respiratorydistress syndrome, allergies, ankylosing spondylitis, amyloidosis,anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunethyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermaldystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosisfetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis,hypereosinophilia, irritable bowel syndrome, multiple sclerosis,myasthenia gravis, myocardial or pericardial inflammation,osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis,Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic anaphylaxis, systemic lupus erythematosus, systemicsclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wernersyndrome, complications of cancer, hemodialysis, and extracorporealcirculation, viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections, and trauma; a neurological disorder such asepilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,Alzheimer's disease, Pick's disease, Huntington's disease, dementia,Parkinson's disease and other extrapyramidal disorders, amyotrophiclateral sclerosis and other motor neuron disorders, progressive neuralmuscular atrophy, retinitis pigmentosa, hereditary ataxias, multiplesclerosis and other demyelinating diseases, bacterial and viralmeningitis, brain abscess, subdural empyema, epidural abscess,suppurative intracranial thrombophlebitis, myelitis and radiculitis,viral central nervous system disease, prion diseases including kuru,Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome,fatal familial insomnia, nutritional and metabolic diseases of thenervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; a disorder affecting growth anddevelopment such as actinic keratosis, arteriosclerosis,atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissuedisease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,polycythemia vera, psoriasis, primary thrombocythemia, renal tubularacidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenneand Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGRsyndrome (Wilms' tumor, aniridia, genitourinary abnormalities, andmental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenhardschorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, and sensorineuralhearing loss; a lipid disorder such as fatty liver, cholestasis, primarybiliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferasedeficiency, myoadenylate deaminase deficiency, hypertriglyceridemia,lipid storage disorders such Fabry's disease, Gaucher's disease,Niemann-Pick's disease, metachromatic leukodystrophy,adrenoleukodystrophy, GM₂ gangliosidosis, and ceroid lipofuscinosis,abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetesmellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminatedfat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimalchange disease, lipomas, atherosclerosis, hypercholesterolemia,hypercholesterolemia with hypertriglyceridemia, primaryhypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease,lecithin:cholesterol acyltransferase deficiency, cerebrotendinousxanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease,Sandhoff's disease, hyperlipidemia, hyperlipenia, lipid myopathies, andobesity; and a cell proliferative disorder such as actinic keratosis,arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixedconnective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnalhemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia,and cancers including adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, uterus, leukemias such asmultiple myeloma, and lymphomas such as Hodgkin's disease.

In another embodiment, a vector capable of expressing KPP or a fragmentor derivative thereof may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofKPP including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantiallypurified KPP in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associatedwith decreased expression or activity of KPP including, but not limitedto, those provided above.

In still another embodiment, an agonist which modulates the activity ofKPP may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of KPP including, butnot limited to, those listed above.

In a further embodiment, an antagonist of KPP may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of KPP. Examples of such disorders include, butare not limited to, those cardiovascular diseases, immune systemdisorders, neurological disorders, disorders affecting growth anddevelopment, lipid disorders, cell proliferative disorders, and cancersdescribed above. In one aspect, an antibody which specifically binds KPPmay be used directly as an antagonist or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to cells ortissues which express KPP.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding KPP may be administered to a subject to treat orprevent a disorder associated with increased expression or activity ofKPP including, but not limited to, those described above.

In other embodiments, any protein, agonist, antagonist, antibody,complementary sequence, or vector embodiments may be administered incombination with other appropriate therapeutic agents. Selection of theappropriate agents for use in combination therapy may be made by one ofordinary skill in the art, according to conventional pharmaceuticalprinciples. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of KPP may be produced using methods which are generallyknown in the art. In particular, purified KPP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind KPP. Antibodies to KPP may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. In an embodiment, neutralizing antibodies (i.e.,those which inhibit dimer formation) can be used therapeutically. Singlechain antibodies (e.g., from camels or llamas) may be potent enzymeinhibitors and may have application in the design of peptide mimetics,and in the development of immuno-adsorbents and biosensors (Muyldermans,S. (2001) J. Biotechnol. 74:277-302).

For the production of antibodies, various hosts including goats,rabbits, rats, mice, camels, dromedaries, llamas, humans, and others maybe immunized by injection with KPP or with any fragment or oligopeptidethereof which has immunogenic properties. Depending on the host species,various adjuvants may be used to increase immunological response. Suchadjuvants include, but are not limited to, Freund's, mineral gels suchas aluminum hydroxide, and surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Gurin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to KPP have an amino acid sequence consisting of atleast about 5 amino acids, and generally will consist of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are substantially identical to a portion of theamino acid sequence of the natural protein, Short stretches of KPP aminoacids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to KPP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. USA 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell.Biol. 62:109-120).

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used (Morrison, S. L. et al. (1984)Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al. (1984)Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceKPP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837;Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for KPP may alsobe generated. For example, such fragments include, but are not limitedto, F(ab′)₂ fragments produced by pepsin digestion of the antibodymolecule and Fab fragments generated by reducing the disulfide bridgesof the F(ab)₂ fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse, W. D. et al. (1989)Science 246:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between KPP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering KPP epitopes is generally used, but a competitivebinding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for KPP. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of KPP-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple KPP epitopes, represents the average affinity,or avidity, of the antibodies for KPP. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular KPP epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theKPP-antibody complex must withstand rigorous manipulations. Low-affinityantibody preparations with K_(a) ranging from about 10⁶ to 10⁷ L/moleare preferred for use in immunopurification and similar procedures whichultimately require dissociation of KPP, preferably in active form, fromthe antibody (Catty, D. (1988) Antibodies, Volume I: A PracticalApproach, IRL Press, Washington D.C.; Liddell, J. E. and A. Cryer (1991)A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New YorkN.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is generally employed in proceduresrequiring precipitation of KPP-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable (Catty, supra; Coligan et al., supra).

In another embodiment of the invention, polynucleotides encoding KPP, orany fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, modifications of gene expression can beachieved by designing complementary sequences or antisense molecules(DNA, RNA, PNA, or modified oligonucleotides) to the coding orregulatory regions of the gene encoding KPP. Such technology is wellknown in the art, and antisense oligonucleotides or larger fragments canbe designed from various locations along the coding or control regionsof sequences encoding KPP (Agrawal, S., ed. (1996) AntisenseTherapeutics, Humana Press, Totawa N.J.).

In therapeutic use, any gene delivery system suitable for introductionof the antisense sequences into appropriate target cells can be used.Antisense sequences can be delivered intracellularly in the form of anexpression plasmid which, upon transcription, produces a sequencecomplementary to at least a portion of the cellular sequence encodingthe target protein (Slater, J. E. et al. (1998) J. Allergy Clin. Immunol102:469-475; Scanlon, K. J. et al. (1995) FASEB J. 9:1288-1296).Antisense sequences can also be introduced intracellularly through theuse of viral vectors, such as retrovirus and adeno-associated virusvectors (Miller, A. D. (1990) Blood 76:271-278; Ausubel et al., supra;Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347). Othergene delivery mechanisms include liposome-derived systems, artificialviral envelopes, and other systems known in the art (Rossi, J. J. (1995)Br. Med. Bull. 51:217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.87:1308-1315; Morris, M. C. et al. (1997) Nucleic Acids Res.25:2730-2736).

In another embodiment of the invention, polynucleotides encoding KPP maybe used for somatic or germline gene therapy. Gene therapy may beperformed to (i) correct a genetic deficiency (e.g., in the cases ofsevere combined immunodeficiency (SCID)-X1 disease characterized byX-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science288:669-672), severe combined immunodeficiency syndrome associated withan inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al.(1995) Science 270:475-480; Bordignon, C. et al. (1995) Science270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216;Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G.et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familialhypercholesterolemia, and hemophilia resulting from Factor VIII orFactor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express aconditionally lethal gene product (e.g., in the case of cancers whichresult from unregulated cell proliferation), or (iii) express a proteinwhich affords protection against intracellular parasites (e.g., againsthuman retroviruses, such as human immunodeficiency virus (HIV)(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996)Proc. Natl. Acad. Sci. USA 93:11395-11399), hepatitis B or C virus (HBV,HCV); fungal parasites, such as Candida albicans and Paracoccidioidesbrasiliensis; and protozoan parasites such as Plasmodium falciparum andTrypanosoma cruzi). In the case where a genetic deficiency in KPPexpression or regulation causes disease, the expression of KPP from anappropriate population of transduced cells may alleviate the clinicalmanifestations caused by the genetic deficiency.

In a further embodiment of the invention, diseases or disorders causedby deficiencies in KPP are treated by constructing mammalian expressionvectors encoding KPP and introducing these vectors by mechanical meansinto KPP-deficient cells. Mechanical transfer technologies for use withcells in vivo or ex vitro include (i) direct DNA microinjection intoindividual cells, (ii) ballistic gold particle delivery, (iii)liposome-mediated transfection, (iv) receptor-mediated gene transfer,and (v) the use of DNA transposons (Morgan, R. A. and W. F. Anderson(1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) Cell 91:501-510;Boulay, J.-L. and H. Rézipon (1998) Curr. Opin. Biotechnol. 9:445-450).

Expression vectors that may be effective for the expression of KPPinclude, but are not limited to, the PcDNA 3.1, EPITAG, PRCCMV2, PREP,PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT,PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.), and PTET-OFF,PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (BD Clontech, Palo Alto Calif.). KPPmay be expressed using (i) a constitutively active promoter, (e.g., fromcytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidinekinase (TK), or β-actin genes), (ii) an inducible promoter (e.g., thetetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin.Biotechnol. 9:451-456), commercially available in the T-REX plasmid(Invitrogen)); the ecdysone-inducible promoter (available in theplasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin induciblepromoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V.and H. M. Blau, supra)), or (iii) a tissue-specific promoter or thenative promoter of the endogenous gene encoding KPP from a normalindividual.

Commercially available liposome transformation kits (e.g., the PERFECTLIPID TRANSFECTION KIT, available from Invitrogen) allow one withordinary skill in the art to deliver polynucleotides to target cells inculture and require minimal effort to optimize experimental parameters.In the alternative, transformation is performed using the calciumphosphate method (Graham, F. L. and A. J. Eb (1973) Virology52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J.1:841-845). The introduction of DNA to primary cells requiresmodification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused bygenetic defects with respect to KPP expression are treated byconstructing a retrovirus vector consisting of (i) the polynucleotideencoding KPP under the control of an independent promoter or theretrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNApackaging signals, and (iii) a Rev-responsive element (RRE) along withadditional retrovirus cis-acting RNA sequences and coding sequencesrequired for efficient vector propagation. Retrovirus vectors (e.g., PFBand PFBNEO) are commercially available (Stratagene) and are based onpublished data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA92:6733-6737), incorporated by reference herein. The vector ispropagated in an appropriate vector producing cell line (VPCL) thatexpresses an envelope gene with a tropism for receptors on the targetcells or a promiscuous envelope protein such as VSVg (Armentano, D. etal. (1987) J. Viral. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol.61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Viral.62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey,R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 toRigg (“Method for obtaining retrovirus packaging cell lines producinghigh transducing efficiency retroviral supernatant”) discloses a methodfor obtaining retrovirus packaging cell lines and is hereby incorporatedby reference. Propagation of retrovirus vectors, transduction of apopulation of cells (e.g., CD4⁺ T-cells), and the return of transducedcells to a patient are procedures well known to persons skilled in theart of gene therapy and have been well documented (Ranga, U. et al.(1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood89:2259-2267; Bonyhadi, M. L. (1997) J. Viral. 71:4707-4716; Ranga, U.et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)Blood 89:2283-2290).

In an embodiment, an adenovirus-based gene therapy delivery system isused to deliver polynucleotides encoding KPP to cells which have one ormore genetic abnormalities with respect to the expression of KPP. Theconstruction and packaging of adenovirus-based vectors are well known tothose with ordinary skill in the art. Replication defective adenovirusvectors have proven to be versatile for importing genes encodingimmunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially usefuladenoviral vectors are described in U.S. Pat. No. 5,707,618 to Armentano(“Adenovirus vectors for gene therapy”), hereby incorporated byreference. For adenoviral vectors, see also Antinozzi, P. A. et al.(1999; Annu. Rev. Nutr. 19:511-544) and Verma, I. M. and N. Somia (1997;Nature 18:389:239-242).

In another embodiment, a herpes-based, gene therapy delivery system isused to deliver polynucleotides encoding KPP to target cells which haveone or more genetic abnormalities with respect to the expression of KPP.The use of herpes simplex virus (HSV)-based vectors may be especiallyvaluable for introducing KPP to cells of the central nervous system, forwhich RSV has a tropism. The construction and packaging of herpes basedvectors are well known to those with ordinary skill in the art. Areplication-competent herpes simplex virus (HSV) type 1-based vector hasbeen used to deliver a reporter gene to the eyes of primates (Liu, X. etal. (1999) Exp. Eye Res. 169:385-395). The construction of a HSV-1 virusvector has also been disclosed in detail in U.S. Pat. No. 5,804,413 toDeLuca (“Herpes simplex virus strains for gene transfer”), which ishereby incorporated by reference. U.S. Pat. No. 5,804,413 teaches theuse of recombinant HSV d92 which consists of a genome containing atleast one exogenous gene to be transferred to a cell under the controlof the appropriate promoter for purposes including human gene therapy.Also taught by this patent are the construction and use of recombinantHSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see alsoGoins, W. F. et al. (1999; J. Virol. 73:519-532) and Xu, H. et al.(1994; Dev. Biol. 163:152-161). The manipulation of cloned herpesvirussequences, the generation of recombinant virus following thetransfection of multiple plasmids containing different segments of thelarge herpesvirus genomes, the growth and propagation of herpesvirus,and the infection of cells with herpesvirus are techniques well known tothose of ordinary skill in the art.

In another embodiment, an alphavirus (positive, single-stranded RNAvirus) vector is used to deliver polynucleotides encoding KPP to targetcells. The biology of the prototypic alphavirus, Semliki Forest Virus(SFV), has been studied extensively and gene transfer vectors have beenbased on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin.Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomicRNA is generated that normally encodes the viral capsid proteins. Thissubgenomic RNA replicates to higher levels than the full length genomicRNA, resulting in the overproduction of capsid proteins relative to theviral proteins with enzymatic activity (e.g., protease and polymerase).Similarly, inserting the coding sequence for KPP into the alphavirusgenome in place of the capsid-coding region results in the production ofa large number of KPP-coding RNAs and the synthesis of high levels ofKPP in vector transduced cells. While alphavirus infection is typicallyassociated with cell lysis within a few days, the ability to establish apersistent infection in hamster normal kidney cells (BHK-21) with avariant of Sindbis virus (SIN) indicates that the lytic replication ofalphaviruses can be altered to suit the needs of the gene therapyapplication (Dryga, S. A. et al. (1997) Virology 228:74-83). The widehost range of alphaviruses will allow the introduction of KPP into avariety of cell types. The specific transduction of a subset of cells ina population may require the sorting of cells prior to transduction. Themethods of manipulating infectious cDNA clones of alphaviruses,performing alphavirus cDNA and RNA transfections, and performingalphavirus infections, are well known to those with ordinary skill inthe art.

Oligonucleotides derived from the transcription initiation site, e.g.,between about positions −10 and +10 from the start site, may also beemployed to inhibit gene expression. Similarly, inhibition can beachieved using triple helix base-pairing methodology. Triple helixpairing is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177).A complementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of RNA moleculesencoding KPP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes may be preparedby any method known in the art for the synthesis of nucleic acidmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA molecules encoding KPP. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize complementary RNA, constitutively or inducibly, can beintroduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytosine, guanine, thymine, anduracil which are not as easily recognized by endogenous endonucleases.

In other embodiments of the invention, the expression of one or moreselected polynucleotides of the present invention can be altered,inhibited, decreased, or silenced using RNA interference (RNAi) orpost-transcriptional gene silencing (PTGS) methods known in the art.RNAi is a post-transcriptional mode of gene silencing in whichdouble-stranded RNA (dsRNA) introduced into a targeted cell specificallysuppresses the expression of the homologous gene (i.e., the gene bearingthe sequence complementary to the dsRNA). This effectively knocks out orsubstantially reduces the expression of the targeted gene. PTGS can alsobe accomplished by use of DNA or DNA fragments as well. RNAi methods aredescribed by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T.(2000; Nature 404:804-808). PTGS can also be initiated by introductionof a complementary segment of DNA into the selected tissue using genedelivery and/or viral vector delivery methods described herein or knownin the art.

RNAi can be induced in mammalian cells by the use of small interferingRNA also known as siRNA. siRNA are shorter segments of dsRNA (typicallyabout 21 to 23 nucleotides in length) that result in vivo from cleavageof introduced dsRNA by the action of an endogenous ribonuclease siRNAappear to be the mediators of the RNAi effect in mammals. The mosteffective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3′overhangs. The use of siRNA for inducing RNAi in mammalian cells isdescribed by Elbasbir, S. M. et al. (2001; Nature 411:494-498).

siRNA can be generated indirectly by introduction of dsRNA into thetargeted cell. Alternatively, siRNA can be synthesized directly andintroduced into a cell by transfection methods and agents describedherein or known in the art (such as liposome-mediated transfection,viral vector methods, or other polynucleotide delivery/introductorymethods). Suitable siRNAs can be selected by examining a transcript ofthe target polynucleotide (e.g., mRNA) for nucleotide sequencesdownstream from the AUG start codon and recording the occurrence of eachnucleotide and the 3′ adjacent 19 to 23 nucleotides as potential siRNAtarget sites, with sequences having a 21 nucleotide length beingpreferred. Regions to be avoided for target siRNA sites include the 5′and 3′ untranslated regions (UTRs) and regions near the start codon(within 75 bases), as these may be richer in regulatory protein bindingsites. UTR-binding proteins and/or translation initiation complexes mayinterfere with binding of the siRNP endonuclease complex. The selectedtarget sites for siRNA can then be compared to the appropriate genomedatabase (e.g., human, etc.) using BLAST or other sequence comparisonalgorithms known in the art. Target sequences with significant homologyto other coding sequences can be eliminated from consideration. Theselected siRNAs can be produced by chemical synthesis methods known inthe art or by in vitro transcription using commercially availablemethods and kits such as the SILENCER siRNA construction kit (Ambion,Austin Tex.).

In alternative embodiments, long-term gene silencing and/or RNAi effectscan be induced in selected tissue using expression vectors thatcontinuously express siRNA. This can be accomplished using expressionvectors that are engineered to express hairpin RNAs (shRNAs) usingmethods known in the art (see, e.g., Brummelkamp, T. R. et al. (2002)Science 296:550-553; and Paddison, P. J. et al. (2002) Genes Dev.16:948-958). In these and related embodiments, shRNAs can be deliveredto target cells using expression vectors known in the art. An example ofa suitable expression vector for delivery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion). Once delivered to the target tissue,shRNAs are processed in vivo into siRNA-like molecules capable ofcarrying out gene-specific silencing.

In various embodiments, the expression levels of genes targeted by RNAior PTGS methods can be determined by assays for mRNA and/or proteinanalysis. Expression levels of the mRNA of a targeted gene can bedetermined, for example, by northern analysis methods using theNORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; byreal time PCR methods; and by other RNA/polynucleotide assays known inthe art or described herein. Expression levels of the protein encoded bythe targeted gene can be determined, for example, by microarray methods;by polyacrylamide gel electrophoresis; and by Western analysis usingstandard techniques known in the art.

An additional embodiment of the invention encompasses a method forscreening for a compound which is effective in altering expression of apolynucleotide encoding KPP. Compounds which may be effective inaltering expression of a specific polynucleotide may include, but arenot limited to, oligonucleotides, antisense oligonucleotides, triplehelix-forming oligonucleotides, transcription factors and otherpolypeptide transcriptional regulators, and non-macromolecular chemicalentities which are capable of interacting with specific polynucleotidesequences. Effective compounds may alter polynucleotide expression byacting as either inhibitors or promoters of polynucleotide expression.Thus, in the treatment of disorders associated with increased KPPexpression or activity, a compound which specifically inhibitsexpression of the polynucleotide encoding KPP may be therapeuticallyuseful, and in the treatment of disorders associated with decreased KPPexpression or activity, a compound which specifically promotesexpression of the polynucleotide encoding KPP may be therapeuticallyuseful.

In various embodiments, one or more test compounds may be screened foreffectiveness in altering expression of a specific polynucleotide. Atest compound may be obtained by any method commonly known in the art,including chemical modification of a compound known to be effective inaltering polynucleotide expression; selection from an existing,commercially-available or proprietary library of naturally-occurring ornon-natural chemical compounds; rational design of a compound based onchemical and/or structural properties of the target polynucleotide; andselection from a library of chemical compounds created combinatoriallyor randomly. A sample comprising a polynucleotide encoding KPP isexposed to at least one test compound thus obtained. The sample maycomprise, for example, an intact or permeabilized cell, or an in vitrocell-free or reconstituted biochemical system. Alterations in theexpression of a polynucleotide encoding KPP are assayed by any methodcommonly known in the art. Typically, the expression of a specificnucleotide is detected by hybridization with a probe having a nucleotidesequence complementary to the sequence of the polynucleotide encodingKPP. The amount of hybridization may be quantified, thus forming thebasis for a comparison of the expression of the polynucleotide both withand without exposure to one or more test compounds. Detection of achange in the expression of a polynucleotide exposed to a test compoundindicates that the test compound is effective in altering the expressionof the polynucleotide. A screen for a compound effective in alteringexpression of a specific polynucleotide can be carried out, for example,using a Schizosaccharomyces pombe gene expression system (Atkins, D. etal. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) NucleicAcids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L.et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particularembodiment of the present invention involves screening a combinatoriallibrary of oligonucleotides (such as deoxyribonucleotides,ribonucleotides, peptide nucleic acids, and modified oligonucleotides)for antisense activity against a specific polynucleotide sequence(Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. etal. (2000) U.S. Pat. No. 6,022,691).

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art (Goldman, C. K. et al. (1997) Nat. Biotechnol.15:462-466).

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such ashumans, dogs, cats, cows, horses, rabbits, and monkeys.

An additional embodiment of the invention relates to the administrationof a composition which generally comprises an active ingredientformulated with a pharmaceutically acceptable excipient. Excipients mayinclude, for example, sugars, starches, celluloses, gums, and proteins.Various formulations are commonly known and are thoroughly discussed inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.). Such compositions may consist of KPP,antibodies to KPP, and mimetics, agonists, antagonists, or inhibitors ofKPP.

In various embodiments, the compositions described herein, such aspharmaceutical compositions, may be administered by any number of routesincluding, but not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal,enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid ordry powder form. These compositions are generally aerosolizedimmediately prior to inhalation by the patient. In the case of smallmolecules (e.g. traditional low molecular weight organic drugs), aerosoldelivery of fast-acting formulations is well-known in the art. In thecase of macromolecules (e.g. larger peptides and proteins), recentdevelopments in the field of pulmonary delivery via the alveolar regionof the lung have enabled the practical delivery of drugs such as insulinto blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No.5,997,848). Pulmonary delivery allows administration without needleinjection, and obviates the need for potentially toxic penetrationenhancers.

Compositions suitable for use in the invention include compositionswherein the active ingredients are contained in an effective amount toachieve the intended purpose. The determination of an effective dose iswell within the capability of those skilled in the art.

Specialized forms of compositions may be prepared for directintracellular delivery of macromolecules comprising KPP or fragmentsthereof. For example, liposome preparations containing acell-impermeable macromolecule may promote cell fusion and intracellulardelivery of the macromolecule. Alternatively, KPP or a fragment thereofmay be joined to a short cationic N-terminal portion from the HIV Tat-1protein. Fusion proteins thus generated have been found to transduceinto the cells of all tissues, including the brain, in a mouse modelsystem (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example KPP or fragments thereof, antibodies of KPP, andagonists, antagonists or inhibitors of KPP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD₅₀/ED₅₀ ratio. Compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies are used to formulate a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that includes the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, the sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.

Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostic

In another embodiment, antibodies which specifically bind KPP may beused for the diagnosis of disorders characterized by expression of KPP,or in assays to monitor patients being treated with KPP or agonists,antagonists, or inhibitors of KPP. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for KPP include methods which utilizethe antibody and a label to detect KPP in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring KPP, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of KPP expression. Normal or standard values for KPPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, for example, human subjects, withantibodies to KPP under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, such as photometric means. Quantities of KPP expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, polynucleotides encoding KPP maybe used for diagnostic purposes. The polynucleotides which may be usedinclude oligonucleotides, complementary RNA and DNA molecules, and PNAs.The polynucleotides may be used to detect and quantify gene expressionin biopsied tissues in which expression of KPP may be correlated withdisease. The diagnostic assay may be used to determine absence,presence, and excess expression of KPP, and to monitor regulation of KPPlevels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotides, including genomic sequences, encoding KPP orclosely related molecules may be used to identify nucleic acid sequenceswhich encode KPP. The specificity of the probe, whether it is made froma highly specific region, e.g., the 5′ regulatory region, or from a lessspecific region, e.g., a conserved motif, and the stringency of thehybridization or amplification will determine whether the probeidentifies only naturally occurring sequences encoding KPP, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, and mayhave at least 50% sequence identity to any of the KPP encodingsequences. The hybridization probes of the subject invention may be DNAor RNA and may be derived from the sequence of SEQ ID NO:44-86 or fromgenomic sequences including promoters, enhancers, and introns of the KPPgene.

Means for producing specific hybridization probes for polynucleotidesencoding KPP include the cloning of polynucleotides encoding KPP or KPPderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotides encoding KPP may be used for the diagnosis of disordersassociated with expression of KPP. Examples of such disorders include,but are not limited to, a cardiovascular disease such as arteriovenousfistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease,aneurysms, arterial dissections, varicose veins, thrombophlebitis andphlebothrombosis, vascular tumors, and complications of thrombolysis,balloon angioplasty, vascular replacement, and coronary artery bypassgraft surgery, congestive heart failure, ischemic heart disease, anginapectoris, myocardial infarction, hypertensive heart disease,degenerative valvular heart disease, calcific aortic valve stenosis,congenitally bicuspid aortic valve, mitral annular calcification, mitralvalve prolapse, rheumatic fever and rheumatic heart disease, infectiveendocarditis, nonbacterial thrombotic endocarditis, endocarditis ofsystemic lupus erythematosus, carcinoid heart disease, cardiomyopathy,myocarditis, pericarditis, neoplastic heart disease, congenital heartdisease, and complications of cardiac transplantation, congenital lunganomalies, atelectasis, pulmonary congestion and edema, pulmonaryembolism, pulmonary hemorrhage, pulmonary infarction, pulmonaryhypertension, vascular sclerosis, obstructive pulmonary disease,restrictive pulmonary disease, chronic obstructive pulmonary disease,emphysema, chronic bronchitis, bronchial asthma, bronchiectasis,bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess,pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophiliabronchiolitis obliterans-organizing pneumonia, diffuse pulmonaryhemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonaryhemosiderosis, pulmonary involvement in collagen-vascular disorders,pulmonary alveolar proteinosis, lung tumors, inflammatory andnoninflammatory pleural effusions; pneumothorax, pleural tumors,drug-induced lung disease, radiation-induced lung disease, andcomplications of lung transplantation; an immune system disorder such asacquired immunodeficiency syndrome (AIDS), Addison's disease, adultrespiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma; aneurological disorder such as epilepsy, ischemic cerebrovasculardisease, stroke, cerebral neoplasms, Alzheimer's disease, Pick'sdisease, Huntington's disease, dementia, Parkinson's disease and otherextrapyramidal disorders, amyotrophic lateral sclerosis and other motorneuron disorders, progressive neural muscular atrophy, retinitispigmentosa, hereditary ataxias, multiple sclerosis and otherdemyelinating diseases, bacterial and viral meningitis, brain abscess,subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease, prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; a disorder affecting growth anddevelopment such as actinic keratosis, arteriosclerosis,atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissuedisease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,polycythemia vera, psoriasis, primary thrombocythemia, renal tubularacidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenneand Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGRsyndrome (Wilms' tumor, aniridia, genitourinary abnormalities, andmental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, and sensorineuralhearing loss; a lipid disorder such as fatty liver, cholestasis, primarybiliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferasedeficiency, myoadenylate deaminase deficiency, hypertriglyceridemia,lipid storage disorders such Fabry's disease, Gaucher's disease,Niemann-Pick's disease, metachromatic leukodystrophy,adrenoleukodystrophy, GM₂ gangliosidosis, and ceroid lipofuscinosis,abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetesmellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminatedfat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimalchange disease, lipomas, atherosclerosis, hypercholesterolemia,hypercholesterolemia with hypertriglyceridemia, primaryhypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease,lecithin:cholesterol acyltransferase deficiency, cerebrotendinousxanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease,Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, andobesity; and a cell proliferative disorder such as actinic keratosis,arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixedconnective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnalhemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia,and cancers including adenocarcinoma, leukemia, lymphoma, melanoma,myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of theadrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, uterus, leukemias such asmultiple myeloma, and lymphomas such as Hodgkin's disease.Polynucleotides encoding KPP may be used in Southern or northernanalysis, dot blot, or other membrane-based technologies; in PCRtechnologies; in dipstick, pin, and multiformat ELISA-like assays; andin microarrays utilizing fluids or tissues from patients to detectaltered KPP expression. Such qualitative or quantitative methods arewell known in the art.

In a particular embodiment, polynucleotides encoding KPP may be used inassays that detect the presence of associated disorders, particularlythose mentioned above. Polynucleotides complementary to sequencesencoding KPP may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantified and compared with astandard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of polynucleotides encoding KPP in the sampleindicates the presence of the associated disorder. Such assays may alsobe used to evaluate the efficacy of a particular therapeutic treatmentregimen in animal studies, in clinical trials, or to monitor thetreatment of an individual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of KPP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding KPP, under conditions suitablefor hybridization or amplification. Standard hybridization may bequantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or overexpressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier, thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding KPP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding KPP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding KPP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantification of closely related DNA or RNA sequences.

In a particular aspect, oligonucleotide primers derived frompolynucleotides encoding KPP may be used to detect single nucleotidepolymorphisms (SNPs). SNPs are substitutions, insertions and deletionsthat are a frequent cause of inherited or acquired genetic disease inhumans. Methods of SNP detection include, but are not limited to,single-stranded conformation polymorphism (SSCP) and fluorescent SSCP(fSSCP) methods. In SSCP, oligonucleotide primers derived frompolynucleotides encoding KPP are used to amplify DNA using thepolymerase chain reaction (PCR). The DNA may be derived, for example,from diseased or normal tissue, biopsy samples, bodily fluids, and thelike. SNPs in the DNA cause differences in the secondary and tertiarystructures of PCR products in single-stranded form, and thesedifferences are detectable using gel electrophoresis in non-denaturinggels. In fSCCP, the oligonucleotide primers are fluorescently labeled,which allows detection of the amplimers in high-throughput equipmentsuch as DNA sequencing machines. Additionally, sequence databaseanalysis methods, termed in silico SNP (isSNP), are capable ofidentifying polymorphisms by comparing the sequence of individualoverlapping DNA fragments which assemble into a common consensussequence. These computer-based methods filter out sequence variationsdue to laboratory preparation of DNA and sequencing errors usingstatistical models and automated analyses of DNA sequence chromatograms.In the alternative, SNPs may be detected and characterized by massspectrometry using, for example, the high throughput MASSARRAY system(Sequenom, Inc., San Diego Calif.).

SNPs may be used to study the genetic basis of human disease. Forexample, at least 16 common SNPs have been associated withnon-insulin-dependent diabetes mellitus. SNPs are also useful forexamining differences in disease outcomes in monogenic disorders, suchas cystic fibrosis, sickle cell anemia, or chronic granulomatousdisease. For example, variants in the mannose-binding lectin, MBL2, havebeen shown to be correlated with deleterious pulmonary outcomes incystic fibrosis. SNPs also have utility in pharmacogenomics, theidentification of genetic variants that influence a patient's responseto a drug, such as life-threatening toxicity. For example, a variationin N-acetyl transferase is associated with a high incidence ofperipheral neuropathy in response to the anti-tuberculosis drugisoniazid, while a variation in the core promoter of the ALOX5 generesults in diminished clinical response to treatment with an anti-asthmadrug that targets the 5-lipoxygenase pathway. Analysis of thedistribution of SNPs in different populations is useful forinvestigating genetic drift, mutation, recombination, and selection, aswell as for tracing the origins of populations and their migrations(Taylor, J. G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. andZ. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr.Opin. Neurobiol. 11:637-641).

Methods which may also be used to quantify the expression of KPP includeradiolabeling or biotinylating nucleotides, coamplification of a controlnucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al.(1993) Anal. Biochem. 212:229-236). The speed of quantitation ofmultiple samples may be accelerated by running the assay in ahigh-throughput format where the oligomer or polynucleotide of interestis presented in various dilutions and a spectrophotometric orcolorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotides described herein may be used as elementson a microarray. The microarray can be used in transcript imagingtechniques which monitor the relative expression levels of large numbersof genes simultaneously as described below. The microarray may also beused to identify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, to monitorprogression/regression of disease as a function of gene expression, andto develop and monitor the activities of therapeutic agents in thetreatment of disease. In particular, this information may be used todevelop a pharmacogenomic profile of a patient in order to select themost appropriate and effective treatment regimen for that patient. Forexample, therapeutic agents which are highly effective and display thefewest side effects may be selected for a patient based on his/herpharmacogenomic profile.

In another embodiment, KPP, fragments of KPP, or antibodies specific forKPP may be used as elements on a microarray. The microarray may be usedto monitor or measure protein-protein interactions, drug-targetinteractions, and gene expression profiles, as described above.

A particular embodiment relates to the use of the polynucleotides of thepresent invention to generate a transcript image of a tissue or celltype. A transcript image represents the global pattern of geneexpression by a particular tissue or cell type. Global gene expressionpatterns are analyzed by quantifying the number of expressed genes andtheir relative abundance under given conditions and at a given time(Seilhamer et al., “Comparative Gene Transcript Analysis,” U.S. Pat. No.5,840,484; hereby expressly incorporated by reference herein). Thus atranscript image may be generated by hybridizing the polynucleotides ofthe present invention or their complements to the totality oftranscripts or reverse transcripts of a particular tissue or cell type.In one embodiment, the hybridization takes place in high-throughputformat, wherein the polynucleotides of the present invention or theircomplements comprise a subset of a plurality of elements on amicroarray. The resultant transcript image would provide a profile ofgene activity.

Transcript images may be generated using transcripts isolated fromtissues, cell lines, biopsies, or other biological samples. Thetranscript image may thus reflect gene expression in vivo, as in thecase of a tissue or biopsy sample, or in vitro, as in the case of a cellline.

Transcript images which profile the expression of the polynucleotides ofthe present invention may also be used in conjunction with in vitromodel systems and preclinical evaluation of pharmaceuticals, as well astoxicological testing of industrial and naturally-occurringenvironmental compounds. All compounds induce characteristic geneexpression patterns, frequently termed molecular fingerprints ortoxicant signatures, which are indicative of mechanisms of action andtoxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153-159;Steiner, S, and N. L. Anderson (2000) Toxicol. Lett. 112-113:467-471).If a test compound has a signature similar to that of a compound withknown toxicity, it is likely to share those toxic properties. Thesefingerprints or signatures are most useful and refined when they containexpression information from a large number of genes and gene families.Ideally, a genome-wide measurement of expression provides the highestquality signature. Even genes whose expression is not altered by anytested compounds are important as well, as the levels of expression ofthese genes are used to normalize the rest of the expression data. Thenormalization procedure is useful for comparison of expression dataafter treatment with different compounds. While the assignment of genefunction to elements of a toxicant signature aids in interpretation oftoxicity mechanisms, knowledge of gene function is not necessary for thestatistical matching of signatures which leads to prediction of toxicity(see, for example, Press Release 00-02 from the National Institute ofEnvironmental Health Sciences, released Feb. 29, 2000, available atniehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important anddesirable in toxicological screening using toxicant signatures toinclude all expressed gene sequences.

In an embodiment, the toxicity of a test compound can be assessed bytreating a biological sample containing nucleic acids with the testcompound. Nucleic acids that are expressed in the treated biologicalsample are hybridized with one or more probes specific to thepolynucleotides of the present invention, so that transcript levelscorresponding to the polynucleotides of the present invention may bequantified. The transcript levels in the treated biological sample arecompared with levels in an untreated biological sample. Differences inthe transcript levels between the two samples are indicative of a toxicresponse caused by the test compound in the treated sample.

Another embodiment relates to the use of the polypeptides disclosedherein to analyze the proteome of a tissue or cell type. The termproteome refers to the global pattern of protein expression in aparticular tissue or cell type. Each protein component of a proteome canbe subjected individually to further analysis. Proteome expressionpatterns, or profiles, are analyzed by quantifying the number ofexpressed proteins and their relative abundance under given conditionsand at a given time. A profile of a cell's proteome may thus begenerated by separating and analyzing the polypeptides of a particulartissue or cell type. In one embodiment, the separation is achieved usingtwo-dimensional gel electrophoresis, in which proteins from a sample areseparated by isoelectric focusing in the first dimension, and thenaccording to molecular weight by sodium dodecyl sulfate slab gelelectrophoresis in the second dimension (Steiner and Anderson, supra).The proteins are visualized in the gel as discrete and uniquelypositioned spots, typically by staining the gel with an agent such asCoomassie Blue or silver or fluorescent stains. The optical density ofeach protein spot is generally proportional to the level of the proteinin the sample. The optical densities of equivalently positioned proteinspots from different samples, for example, from biological sampleseither treated or untreated with a test compound or therapeutic agent,are compared to identify any changes in protein spot density related tothe treatment. The proteins in the spots are partially sequenced using,for example, standard methods employing chemical or enzymatic cleavagefollowed by mass spectrometry. The identity of the protein in a spot maybe determined by comparing its partial sequence, preferably of at least5 contiguous amino acid residues, to the polypeptide sequences ofinterest. In some cases, further sequence data may be obtained fordefinitive protein identification.

A proteomic profile may also be generated using antibodies specific forKPP to quantify the levels of KPP expression. In one embodiment, theantibodies are used as elements on a microarray, and protein expressionlevels are quantified by contacting the microarray with the sample anddetecting the levels of protein bound to each array element (Lueking, A.et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999)Biotechniques 27:778-788). Detection may be performed by a variety ofmethods known in the art, for example, by reacting the proteins in thesample with a thiol- or amino-reactive fluorescent compound anddetecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful fortoxicological screening, and should be analyzed in parallel withtoxicant signatures at the transcript level. There is a poor correlationbetween transcript and protein abundances for some proteins in sometissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis18:533-537), so proteome toxicant signatures may be useful in theanalysis of compounds which do not significantly affect the transcriptimage, but which alter the proteomic profile. In addition, the analysisof transcripts in body fluids is difficult, due to rapid degradation ofmRNA, so proteomic profiling may be more reliable and informative insuch cases.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins that are expressed in the treated biological sample areseparated so that the amount of each protein can be quantified. Theamount of each protein is compared to the amount of the correspondingprotein in an untreated biological sample. A difference in the amount ofprotein between the two samples is indicative of a toxic response to thetest compound in the treated sample. Individual proteins are identifiedby sequencing the amino acid residues of the individual proteins andcomparing these partial sequences to the polypeptides of the presentinvention.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins from the biological sample are incubated with antibodiesspecific to the polypeptides of the present invention. The amount ofprotein recognized by the antibodies is quantified. The amount ofprotein in the treated biological sample is compared with the amount inan untreated biological sample. A difference in the amount of proteinbetween the two samples is indicative of a toxic response to the testcompound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known inthe art (Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena,M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619;Baldeschweiler et al. (1995) PCT application WO95/25116; Shalon, D. etal. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc.Natl. Acad. Sci. USA 94:2150-2155; Heller, M. J. et al. (1997) U.S. Pat.No. 5,605,662). Various types of microarrays are well known andthoroughly described in Schena, M., ed. (1999; DNA Microarrays: APractical Approach, Oxford University Press, London).

In another embodiment of the invention, nucleic acid sequences encodingKPP may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. Either coding or noncodingsequences may be used, and in some instances, noncoding sequences may bepreferable over coding sequences. For example, conservation of a codingsequence among members of a multi-gene family may potentially causeundesired cross hybridization during chromosomal mapping. The sequencesmay be mapped to a particular chromosome, to a specific region of achromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome cDNA libraries (Harrington, J. J. et al. (1997) Nat.Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet. 7:149-154). Once mapped, the nucleic acidsequences may be used to develop genetic linkage maps, for example,which correlate the inheritance of a disease state with the inheritanceof a particular chromosome region or restriction fragment lengthpolymorphism (RFLP) (Lander, E. S. and D. Botstein (1986) Proc. Natl.Acad. Sci. USA 83:7353-7357).

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers,supra, pp. 965-968). Examples of genetic map data can be found invarious scientific journals or at the Online Mendelian Inheritance inMan (OMIM) World Wide Web site. Correlation between the location of thegene encoding KPP on a physical map and a specific disorder, or apredisposition to a specific disorder, may help define the region of DNAassociated with that disorder and thus may further positional cloningefforts.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the exact chromosomal locus is notknown. This information is valuable to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once the gene or genes responsible for a disease or syndromehave been crudely localized by genetic linkage to a particular genomicregion, e.g., ataxia-telangiectasia to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation (Gatti, R. A. et al. (1988) Nature 336:577-580). Thenucleotide sequence of the instant invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc., among normal, carrier, or affected individuals.

In another embodiment of the invention, KPP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between KPPand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest (Geysen, et al. (1984) PCT application WO84/03564). In thismethod, large numbers of different small test compounds are synthesizedon a solid substrate. The test compounds are reacted with KPP, orfragments thereof, and washed. Bound KPP is then detected by methodswell known in the art. Purified KPP can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding KPP specificallycompete with a test compound for binding KPP. In this manner, antibodiescan be used to detect the presence of any peptide which shares one ormore antigenic determinants with KPP.

In additional embodiments, the nucleotide sequences which encode KPP maybe used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

Without further elaboration, it is believed that one skilled in the aftcan, using the preceding description, utilize the present invention toits fullest extent. The following embodiments are, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever.

The disclosures of all patents, applications, and publications mentionedabove and below, is including U.S. Ser. No. 60/467,491, U.S. Ser. No.60/469,441, U.S. Ser. No. 60/476,408, U.S. Ser. No. 60/494,656, U.S.Ser. No. 60/524,415, and U.S. Ser. No. 60/528,750, are hereby expresslyincorporated by reference.

EXAMPLES I. Construction of cDNA Libraries

Incyte cDNAs are derived from cDNA libraries described in the LIFFSEQdatabase (Incyte, Palo Alto Calif.). Some tissues are homogenized andlysed in guanidinium isothiocyanate, while others are homogenized andlysed in phenol or in a suitable mixture of denaturants, such as TRIZOL(Invitrogen), a monophasic solution of phenol and guanidineisothiocyanate. The resulting lysates are centrifuged over CsCl cushionsor extracted with chloroform. RNA is precipitated from the lysates witheither isopropanol or sodium acetate and ethanol, or by other routinemethods.

Phenol extraction and precipitation of RNA are repeated as necessary toincrease RNA purity. In some cases, RNA is treated with DNase. For mostlibraries, poly(A)+ RNA is isolated using oligo d(T)-coupledparamagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN,Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN).Alternatively, RNA is isolated directly from tissue lysates using otherRNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,Austin Tex.).

In some cases, Stratagene is provided with RNA and constructs thecorresponding cDNA libraries. Otherwise, cDNA is synthesized and cDNAlibraries are constructed with the UNIZAP vector system (Stratagene) orSUPERSCRIPT plasmid system (Invitrogen), using the recommendedprocedures or similar methods known in the art (Ausubel et al., supra,ch. 5). Reverse transcription is initiated using oligo d(T) or randomprimers. Synthetic oligonucleotide adapters are ligated to doublestranded cDNA, and the cDNA is digested with the appropriate restrictionenzyme or enzymes. For most libraries, the cDNA is size-selected(300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4Bcolumn chromatography (Amersham Biosciences) or preparative agarose gelelectrophoresis. cDNAs are ligated into compatible restriction enzymesites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid(Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad Calif.), PcDNA2.1plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid(Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte, Palo AltoCalif.), pRARE (Incyte), or pINCY (Incyte), or derivatives thereof.Recombinant plasmids are transformed into competent E. coli cellsincluding XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5α, DH10B,or ElectroMAX DH10B from Invitrogen.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I are recovered from hostcells by in vivo excision using the UNIZAP vector system (Stratagene) orby cell lysis. Plasmids are purified using at least one of thefollowing: a Magic or WIZARD Minipreps DNA purification system(Promega); an AGTC Miniprep purification kit (Edge Biosystems,Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96plasmid purification kit from QIAGEN. Following precipitation, plasmidsare resuspended in 0.1 ml of distilled water and stored, with or withoutlyophilization, at 4° C.

Alternatively, plasmid DNA is amplified from host cell lysates usingdirect link PCR in a high-throughput format (Rao, V. B. (1994) Anal.Biochem. 216:1-14). Host cell lysis and thermal cycling steps arecarried out in a single reaction mixture. Samples are processed andstored in 384-well plates, and the concentration of amplified plasmidDNA is quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Oreg.) and a FLUOROSKAN II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example II aresequenced as follows. Sequencing reactions are processed using standardmethods or high-throughput instrumentation such as the ABI CATALYST 800(Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJResearch) in conjunction with the HYDRA microdispenser (RobbinsScientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNAsequencing reactions are prepared using reagents provided by AmershamBiosciences or supplied in ABI sequencing kits such as the ABI PRISMBIGDYE Terminator cycle sequencing ready reaction kit (AppliedBiosystems). Electrophoretic separation of cDNA sequencing reactions anddetection of labeled polynucleotides are carried out using the MEGABACE1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or377 sequencing system (Applied Biosystems) in conjunction with standardABI protocols and base calling software; or other sequence analysissystems known in the art. Reading frames within the cDNA sequences areidentified using standard methods (Ausubel et al., supra, ch. 7). Someof the cDNA sequences are selected for extension using the techniquesdisclosed in Example VIII.

Polynucleotide sequences derived from Incyte cDNAs are validated byremoving vector, linker, and poly(A) sequences and by masking ambiguousbases, using algorithms and programs based on BLAST, dynamicprogramming, and dinucleotide nearest neighbor analysis. The Incyte cDNAsequences or translations thereof are then queried against a selectionof public databases such as the GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM;PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus,Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Candida albicans (Incyte, Palo AltoCalif.); hidden Markov model (HMM)-based protein family databases suchas PFAM, INCY, and TIGRFAM (Haft, D. H. et al. (2001) Nucleic Acids Res.29:41-43); and HMM-based protein domain databases such as SMART(Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is aprobabilistic approach which analyzes consensus primary structures ofgene families; see, for example, Eddy, S. R. (1996) Curr. Opin. Struct.Biol. 6:361-365.) The queries are performed using programs based onBLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences are assembledto produce full length polynucleotide sequences. Alternatively, GenBankcDNAs, GenBank ESTs, stitched sequences, stretched sequences, orGenscan-predicted coding sequences (see Examples IV and V) are used toextend Incyte cDNA assemblages to full length. Assembly is performedusing programs based on Phred, Phrap, and Consed, and cDNA assemblagesare screened for open reading frames using programs based on GeneMark,BLAST, and PASTA. The full length polynucleotide sequences aretranslated to derive the corresponding full length polypeptidesequences. Alternatively, a polypeptide may begin at any of themethionine residues of the full length translated polypeptide. Fulllength polypeptide sequences are subsequently analyzed by queryingagainst databases such as the GenBank protein databases (genpept),SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM,Prosite, bidden Markov model (HMM)-based protein family databases suchas PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases suchas SMART. Full length polynucleotide sequences are also analyzed usingMAcDNASIS PRO software (MiraiBio, Alameda Calif.) and LASERGENE software(DNASTAR). Polynucleotide and polypeptide sequence alignments aregenerated using default parameters specified by the CLUSTAL algorithm asincorporated into the MEGALIGN multisequence alignment program(DNASTAR), which also calculates the percent identity between alignedsequences.

Table 7 summarizes tools, programs, and algorithms used for the analysisand assembly of Incyte cDNA and full length sequences and providesapplicable descriptions, references, and threshold parameters. The firstcolumn of Table 7 shows the tools, programs, and algorithms used, thesecond column provides brief descriptions thereof, the third columnpresents appropriate references, all of which are incorporated byreference herein in their entirety, and the fourth column presents,where applicable, the scores, probability values, and other parametersused to evaluate the strength of a match between two sequences (thehigher the score or the lower the probability value, the greater theidentity between two sequences).

The programs described above for the assembly and analysis of fulllength polynucleotide and polypeptide sequences are also used toidentify polynucleotide sequence fragments from SEQ ID NO:44-86.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies are described in Table 4,column 2.

IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative kinases and phosphatases are initially identified by runningthe Genscan gene identification program against public genomic sequencedatabases (e.g., gbpri and gbhtg). Genscan is a general-purpose geneidentification program which analyzes genomic DNA sequences from avariety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol.268:78-94; Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol.8:346-354). The program concatenates predicted exons to form anassembled cDNA sequence extending from a methionine to a stop codon. Theoutput of Genscan is a FASTA database of polynucleotide and polypeptidesequences. The maximum range of sequence for Genscan to analyze at onceis set to 30 kb. To determine which of these Genscan predicted cDNAsequences encode kinases and phosphatases, the encoded polypeptides areanalyzed by querying against PFAM models for kinases and phosphatases.Potential kinases and phosphatases are also identified by homology toIncyte cDNA sequences that have been annotated as kinases andphosphatases. These selected Genscan-predicted sequences are thencompared by BLAST analysis to the genpept and gbpri public databases.Where necessary, the Genscan-predicted sequences are then edited bycomparison to the top BLAST bit from genpept to correct errors in thesequence predicted by Genscan, such as extra or omitted exons. BLASTanalysis is also used to find any Incyte cDNA or public cDNA coverage ofthe Genscan-predicted sequences, thus providing evidence fortranscription. When Incyte cDNA coverage is available, this informationis used to correct or confirm the Genscan predicted sequence. Fulllength polynucleotide sequences are obtained by assemblingGenscan-predicted coding sequences with Incyte cDNA sequences and/orpublic cDNA sequences using the assembly process described in ExampleIII. Alternatively, full length polynucleotide sequences are derivedentirely from edited or unedited Genscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data “Stitched”Sequences

Partial cDNA sequences are extended with exons predicted by the Genscangene identification program described in Example IV. Partial cDNAsassembled as described in Example III are mapped to genomic DNA andparsed into clusters containing related cDNAs and Genscan exonpredictions from one or more genomic sequences. Each cluster is analyzedusing an algorithm based on graph theory and dynamic programming tointegrate cDNA and genomic information, generating possible splicevariants that are subsequently confirmed, edited, or extended to createa full length sequence. Sequence intervals in which the entire length ofthe interval is present on more than one sequence in the cluster areidentified, and intervals thus identified are considered to beequivalent by transitivity. For example, if an interval is present on acDNA and two genomic sequences, then all three intervals are consideredto be equivalent. This process allows unrelated but consecutive genomicsequences to be brought together, bridged by cDNA sequence. Intervalsthus identified are then “stitched” together by the stitching algorithmin the order that they appear along their parent sequences to generatethe longest possible sequence, as well as sequence variants. Linkagesbetween intervals which proceed along one type of parent sequence (cDNAto cDNA or genomic sequence to genomic sequence) are given preferenceover linkages which change parent type (cDNA to genomic sequence). Theresultant stitched sequences are translated and compared by BLASTanalysis to the genpept and gbpri public databases. Incorrect exonspredicted by Genscan are corrected by comparison to the top BLAST hitfrom genpept. Sequences are further extended with additional cDNAsequences, or by inspection of genomic DNA, when necessary.

“Stretched” Sequences

Partial DNA sequences are extended to full length with an algorithmbased on BLAST analysis. First, partial cDNAs assembled as described inExample III are queried against public databases such as the GenBankprimate, rodent, mammalian, vertebrate, and eukaryote databases usingthe BLAST program. The nearest GenBank protein homolog is then comparedby BLAST analysis to either Incyte cDNA sequences or GenScan exonpredicted sequences described in Example IV. A chimeric protein isgenerated by using the resultant high-scoring segment pairs (HSPs) tomap the translated sequences onto the GenBank protein homolog.Insertions or deletions may occur in the chimeric protein with respectto the original GenBank protein homolog. The GenBank protein homolog,the chimeric protein, or both are used as probes to search forhomologous genomic sequences from the public human genome databases.Partial DNA sequences are therefore “stretched” or extended by theaddition of homologous genomic sequences. The resultant stretchedsequences are examined to determine whether they contain a completegene.

VI. Chromosomal Mapping of KPP Encoding Polynucleotides

The sequences used to assemble SEQ ID NO:44-86 are compared withsequences from the Incyte LIFESEQ database and public domain databasesusing BLAST and other implementations of the Smith-Waterman algorithm.Sequences from these databases that matched SEQ ID NO:44-86 areassembled into clusters of contiguous and overlapping sequences usingassembly algorithms such as Phrap (Table 7). Radiation hybrid andgenetic mapping data available from public resources such as theStanford Human Genome Center (SHGC), Whitehead Institute for GenomeResearch (WIGR), and Généthon are used to determine if any of theclustered sequences have been previously mapped. Inclusion of a mappedsequence in a cluster results in the assignment of all sequences of thatcluster, including its particular SEQ ID NO:, to that map location.

Map locations are represented by ranges, or intervals, of humanchromosomes. The map position of an interval, in centiMorgans, ismeasured relative to the terminus of the chromosome's p-arm. (ThecentiMorgan (cM) is a unit of measurement based on recombinationfrequencies between chromosomal markers. On average, 1 cM is roughlyequivalent to 1 megabase (Mb) of DNA in humans, although this can varywidely due to hot and cold spots of recombination.) The cM distances arebased on genetic markers mapped by Généthon which provide boundaries forradiation hybrid markers whose sequences were included in each of theclusters. Human genome maps and other resources available to the public,such as the NCBI “GeneMap'99” World Wide Web site(ncbi.nlm.nih.gov/genemap/), can be employed to determine if previouslyidentified disease genes map within or in proximity to the intervalsindicated above.

VII. Analysis of Polynucleotide Expression

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook and Russell, supra, ch. 7;Ausubel et al., supra, ch. 4).

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in databases such as GenBank or LIFESEQ(Incyte). This analysis is much faster than multiple membrane-basedhybridizations. In addition, the sensitivity of the computer search canbe modified to determine whether any particular match is categorized asexact or similar. The basis of the search is the product score, which isdefined as:

$\frac{{BLAST}\mspace{14mu}{Score} \times {Percent}\mspace{14mu}{Identity}}{5 \times {minimum}\mspace{14mu}\left\{ {{{length}\left( {{Seq}.\mspace{14mu} 1} \right)},\mspace{14mu}{{length}\left( {{Seq}.\mspace{14mu} 2} \right)}} \right\}}$The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. The productscore is a normalized value between 0 and 100, and is calculated asfollows: the BLAST score is multiplied by the percent nucleotideidentity and the product is divided by (5 times the length of theshorter of the two sequences). The BLAST score is calculated byassigning a score of +5 for every base that matches in a high-scoringsegment pair (HSP), and −4 for every mismatch. Two sequences may sharemore than one HSP (separated by gaps). If there is more than one HSP,then the pair with the highest BLAST score is used to calculate theproduct score. The product score represents a balance between fractionaloverlap and quality in a BLAST alignment. For example, a product scoreof 100 is produced only for 100% identity over the entire length of theshorter of the two sequences being compared. A product score of 70 isproduced either by 100% identity and 70% overlap at one end, or by 88%identity and 100% overlap at the other. A product score of 50 isproduced either by 100% identity and 50% overlap at one end, or 79%identity and 100% overlap.

Alternatively, polynucleotides encoding KPP are analyzed with respect tothe tissue sources from which they are derived. For example, some fulllength sequences are assembled, at least in part, with overlappingIncyte cDNA sequences (see Example III). Each cDNA sequence is derivedfrom a cDNA library constructed from a human tissue. Each human tissueis classified into one of the following organ/tissue categories:cardiovascular system; connective tissue; digestive system; embryonicstructures; endocrine system; exocrine glands; genitalia, female;genitalia, male; germ cells; hemic and immune system; liver;musculoskeletal system; nervous system; pancreas; respiratory system;sense organs; skin; stomatognathic system; unclassified/mixed; orurinary tract. The number of libraries in each category is counted anddivided by the total number of libraries across all categories.Similarly, each human tissue is classified into one of the followingdisease/condition categories: cancer, cell line, developmental,inflammation, neurological, trauma, cardiovascular, pooled, and other,and the number of libraries in each category is counted and divided bythe total number of libraries across all categories. The resultingpercentages reflect the tissue- and disease-specific expression of cDNAencoding KPP. cDNA sequences and cDNA library/tissue information arefound in the LIFESEQ database (Incyte, Palo Alto Calif.).

VIII. Extension of KPP Encoding Polynucleotides

Full length polynucleotides are produced by extension of an appropriatefragment of the full length molecule using oligonucleotide primersdesigned from this fragment. One primer is synthesized to initiate 5′extension of the known fragment, and the other primer is synthesized toinitiate 3′ extension of the known fragment. The initial primers aredesigned using OLIGO 4.06 software (National Biosciences), or anotherappropriate program, to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to about 72° C. Any stretch ofnucleotides which would result in hairpin structures and primer-primerdimerizations is avoided.

Selected human cDNA libraries are used to extend the sequence. If morethan one extension is necessary or desired, additional or nested sets ofprimers are designed.

High fidelity amplification is obtained by PCR using methods well knownin the art. PCR is performed in 96-well plates using the PTC-200 thermalcycler (MJ Research, Inc.). The reaction mix contains DNA template, 200nmol of each primer, reaction buffer containing Mg²⁺, (NH₄)₂SO₄, and2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASEenzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with thefollowing parameters for primer pair PCI A and PCI B: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 60° C.; 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C. In the alternative, the parameters for primerpair T7 and SK+ are as follows: Step 1: 94° C., 3 min; Step 2: 94° C.,15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2 min; Step 5: Steps 2,3, and 4 repeated 20 times; Step 6: 68° C., 5 min; Step 7: storage at 4°C.

The concentration of DNA in each well is determined by dispensing 100 μlPICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes,Eugene Oreg.) dissolved in 1×TE and 0.5 μl of undiluted PCR product intoeach well of an opaque fluorimeter plate (Corning Costar, Acton Mass.),allowing the DNA to bind to the reagent. The plate is scanned in aFluoroskan II (Labsystems Oy, Helsinki, Finland) to measure thefluorescence of the sample and to quantify the concentration of DNA. A 5μl to 10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a 1% agarose gel to determine which reactions aresuccessful in extending the sequence.

The extended nucleotides are desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Biosciences). Forshotgun sequencing, the digested nucleotides are separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments are excised, andagar digested with Agar ACE (Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly Mass.) into pUC18 vector(Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) tofill-in restriction site overhangs, and transfected into competent E.coli cells. Transformed cells are selected on antibiotic-containingmedia, and individual colonies are picked and cultured overnight at 37°C. in 384-well plates in LB/2× carb liquid media.

The cells are lysed, and DNA is amplified by PCR using Taq DNApolymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene)with the following parameters: Step 1: 94° C., 3 min; Step 2: 94° C., 15sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5: steps 2, 3,and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7: storage at 4° C.DNA is quantified by PICOGREEN reagent (Molecular Probes) as describedabove. Samples with low DNA recoveries are reamplified using the sameconditions as described above. Samples are diluted with 20%dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energytransfer sequencing Primers and the DYENAMIC DIRECT kit (AmershamBiosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing readyreaction kit (Applied Biosystems).

In like manner, full length polynucleotides are verified using the aboveprocedure or are used to obtain 5′ regulatory sequences using the aboveprocedure along with oligonucleotides designed for such extension, andan appropriate genomic library.

IX. Identification of Single Nucleotide Polymorphisms in KPP EncodingPolynucleotides

Common DNA sequence variants known as single nucleotide polymorphisms(SNPs) are identified in SEQ ID NO:44-86 using the LIFESEQ database(Incyte). Sequences from the same gene are clustered together andassembled as described in Example III, allowing the identification ofall sequence variants in the gene. An algorithm consisting of a seriesof filters is used to distinguish SNPs from other sequence variants.Preliminary filters remove the majority of basecall errors by requiringa minimum Phred quality score of 15, and remove sequence alignmenterrors and errors resulting from improper trimming of vector sequences,chimeras, and splice variants. An automated procedure of advancedchromosome analysis is applied to the original chromatogram files in thevicinity of the putative SNP. Clone error filters use statisticallygenerated algorithms to identify errors introduced during laboratoryprocessing, such as those caused by reverse transcriptase, polymerase,or somatic mutation. Clustering error filters use statisticallygenerated algorithms to identify errors resulting from clustering ofclose homologs or pseudogenes, or due to contamination by non-humansequences. A final set of filters removes duplicates and SNPs found inimmunoglobulins or T-cell receptors.

Certain SNPs are selected for further characterization by massspectrometry using the high throughput MASSARRAY system (Sequenom, Inc.)to analyze allele frequencies at the SNP sites in four different humanpopulations. The Caucasian population comprises 92 individuals (46 male,46 female), including 83 from Utah, four French, three Venezualan, andtwo Amish individuals. The African population comprises 194 individuals(97 male, 97 female), all African Americans. The Hispanic populationcomprises 324 individuals (162 male, 162 female), all Mexican Hispanic.The Asian population comprises 126 individuals (64 male, 62 female) witha reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean,5% Vietnamese, and 8% other Asian. Allele frequencies are first analyzedin the Caucasian population; in some cases those SNPs which show noallelic variance in this population are not further tested in the otherthree populations.

X. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:44-86 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham Biosciences), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Biosciences). An aliquot containing 10⁷counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to NYTRAN PLUS nylon membranes (Schleicher & Schuell, DurhamN.H.). Hybridization is carried out for 16 hours at 40° C. To removenonspecific signals, blots are sequentially washed at room temperatureunder conditions of up to, for example, 0.1× saline sodium citrate and0.5% sodium dodecyl sulfate. Hybridization patterns are visualized usingautoradiography or an alternative imaging means and compared.

XI. Microarrays

The linkage or synthesis of array elements upon a microarray can beachieved utilizing photolithography, piezoelectric printing (ink-jetprinting; see, e.g., Baldeschweiler et al., supra), mechanicalmicrospotting technologies, and derivatives thereof. The substrate ineach of the aforementioned technologies should be uniform and solid witha non-porous surface (Schena, M., ed. (1999) DNA Microarrays: APractical Approach, Oxford University Press, London). Suggestedsubstrates include silicon, silica, glass slides, glass chips, andsilicon wafers. Alternatively, a procedure analogous to a dot or slotblot may also be used to arrange and link elements to the surface of asubstrate using thermal, UV, chemical, or mechanical bonding procedures.A typical array may be produced using available methods and machineswell known to those of ordinary skill in the art and may contain anyappropriate number of elements (Schena, M. et al. (1995) Science270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall,A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).

Full length cDNAs, Expressed Sequence Tags (SSTs), or fragments oroligomers thereof may comprise the elements of the microarray. Fragmentsor oligomers suitable for hybridization can be selected using softwarewell known in the art such as LASERGENE software (DNASTAR). The arrayelements are hybridized with polynucleotides in a biological sample. Thepolynucleotides in the biological sample are conjugated to a fluorescentlabel or other molecular tag for ease of detection. After hybridization,nonhybridized nucleotides from the biological sample are removed, and afluorescence scanner is used to detect hybridization at each arrayelement. Alternatively, laser desorbtion and mass spectrometry may beused for detection of hybridization. The degree of complementarity andthe relative abundance of each polynucleotide which hybridizes to anelement on the microarray may be assessed. In one embodiment, microarraypreparation and usage is described in detail below.

Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guanidiniumthiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT)cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed usingMMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1×first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μMdGTP, 500 AM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5(Amersham Biosciences). The reverse transcription reaction is performedin a 25 ml volume containing 200 ng poly(A)⁺ RNA with GEMBRIGHT kits(Incyte). Specific control poly(A)⁺ RNAs are synthesized by in vitrotranscription from non-coding yeast genomic DNA. After incubation at 37°C. for 2 hr, each reaction sample (one with Cy3 and another with Cy5labeling) is treated with 2.5 ml of 0.5 M sodium hydroxide and incubatedfor 20 minutes at 85° C. to the stop the reaction and degrade the RNA.Samples are purified using two successive CHROMA SPIN 30 gel filtrationspin columns (BD Clontech, Palo Alto Calif.) and after combining, bothreaction samples are ethanol precipitated using 1 ml of glycogen (1mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample isthen dried to completion using a SpeedVAC (Savant Instruments Inc.,Holbrook N.Y.) and resuspended in 14 μl 5×SSC/0.2% SDS.

Microarray Preparation

Sequences of the present invention are used to generate array elements.Each array element is amplified from bacterial cells containing vectorswith cloned cDNA inserts. PCR amplification uses primers complementaryto the vector sequences flanking the cDNA insert. Array elements areamplified in thirty cycles of PCR from an initial quantity of 1-2 ng toa final quantity greater than 5 μg. Amplified array elements are thenpurified using SEPHACRYL-400 (Amersham Biosciences).

Purified array elements are immobilized on polymer-coated glass slides.Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDSand acetone, with extensive distilled water washes between and aftertreatments. Glass slides are etched in 4% hydrofluoric acid (VWRScientific Products Corporation (VWR), West Chester Pa.), washedextensively in distilled water, and coated with 0.05% aminopropyl silane(Sigma-Aldrich, St. Louis Mo.) in 95% ethanol. Coated slides are curedin a 110° C. oven.

Array elements are applied to the coated glass substrate using aprocedure described in U.S. Pat. No. 5,807,522, incorporated herein byreference. 1 μl of the array element DNA, at an average concentration of100 ng/μl, is loaded into the open capillary printing element by ahigh-speed robotic apparatus. The apparatus then deposits about 5 nl ofarray element sample per slide.

Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker(Stratagene). Microarrays are washed at room temperature once in 0.2%SDS and three times in distilled water. Non-specific binding sites areblocked by incubation of microarrays in 0.2% casein in phosphatebuffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at60° C. followed by washes in 0.2% SDS and distilled water as before.

Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2%SDS hybridization buffer. The sample mixture is heated to 65° C. for 5minutes and is aliquoted onto the microarray surface and covered with an1.8 cm² coverslip. The arrays are transferred to a waterproof chamberhaving a cavity just slightly larger than a microscope slide. Thechamber is kept at 100% humidity internally by the addition of 140 μl of5×SSC in a corner of the chamber. The chamber containing the arrays isincubated for about 6.5 hours at 60° C. The arrays are washed for 10 minat 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10minutes each at 45° C. in a second wash buffer (0.1× SSC), and dried.

Detection

Reporter-labeled hybridization complexes are detected with a microscopeequipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., SantaClara Calif.) capable of generating spectral lines at 488 nm forexcitation of Cy3 and at 632 nm for excitation of Cy5. The excitationlaser light is focused on the array using a 20× microscope Objective(Nikon, Inc., Melville N.Y.). The slide containing the array is placedon a computer-controlled X-Y stage on the microscope and raster-scannedpast the objective. The 1.8 cm×1.8 cm array used in the present exampleis scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the twofluorophores sequentially. Emitted light is split, based on wavelength,into two photomultiplier tube detectors (PMT R1477, Hamamatsu PhotonicsSystems, Bridgewater N.J.) corresponding to the two fluorophores.Appropriate filters positioned between the array and the photomultipliertubes are used to filter the signals. The emission maxima of thefluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array istypically scanned twice, one scan per fluorophore using the appropriatefilters at the laser source, although the apparatus is capable ofrecording the spectra from both fluorophores simultaneously.

The sensitivity of the scans is typically calibrated using the signalintensity generated by a cDNA control species added to the samplemixture at a known concentration. A specific location on the arraycontains a complementary DNA sequence, allowing the intensity of thesignal at that location to be correlated with a weight ratio ofhybridizing species of 1:100,000. When two samples from differentsources (e.g., representing test and control cells), each labeled with adifferent fluorophore, are hybridized to a single array for the purposeof identifying genes that are differentially expressed, the calibrationis done by labeling samples of the calibrating cDNA with the twofluorophores and adding identical amounts of each to the hybridizationmixture.

The output of the photomultiplier tube is digitized using a 12-bitRTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc.,Norwood Mass.) installed in an IBM-compatible PC computer. The digitizeddata are displayed as an image where the signal intensity is mappedusing a linear 20-color transformation to a pseudocolor scale rangingfrom blue (low signal) to red (high signal). The data is also analyzedquantitatively. Where two different fluorophores are excited andmeasured simultaneously, the data are first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that thesignal from each spot is centered in each element of the grid. Thefluorescence signal within each element is then integrated to obtain anumerical value corresponding to the average intensity of the signal.The software used for signal analysis is the GEMTOOLS gene expressionanalysis program (Incyte). Array elements that exhibit at least about atwo-fold change in expression, a signal-to-background ratio of at leastabout 2.5, and an element spot size of at least about 40%, areconsidered to be differentially expressed.

Expression

For example, SEQ ID NO:51, SEQ ID NO:53-54, and SEQ ID NO:57 weredifferentially expressed in breast carcinoma cell lines versus a cellline derived from normal breast epithelial tissue as determined bymicroarray analysis. Gene expression profiles of nonmalignant mammaryepithelial cells were compared to gene expression profiles of variousbreast carcinoma lines at different stages of tumor progression. Thecells were grown in defined serum-free H14 medium to 70-80% confluenceprior to RNA harvest. Cell lines compared included: a) HMEC, a primarybreast epithelial cell line isolated from a normal donor, b) MCF-10A, abreast mammary gland cell line isolated from a 36-year-old woman withfibrocystic breast disease, c) MCF7, a nonmalignant breastadenocarcinoma cell line isolated from the pleural effusion of a69-year-old female, d) T-47D, a breast carcinoma cell line isolated froma pleural effusion obtained from a 54-year-old female with aninfiltrating ductal carcinoma of the breast, e) Sk-BR-3, a breastadenocarcinoma cell line isolated from a malignant pleural effusion of a43-year-old female, f) BT-20, a breast carcinoma cell line derived invitro from cells emigrating out of thin slices of the tumor massisolated from a 74-year-old female, g) MDA-nib-231, a breast tumor cellline isolated from the pleural effusion of a 51-year-old female, and h)MDA-mb-435S, a spindle-shaped strain that evolved from the parent line(435) isolated by R. Cailleau from pleural effusion of a 31-year-oldfemale with metastatic, ductal adenocarcinoma of the breast. Expressionof SEQ ID NO:53 was increased at least two-fold in MCF7 cells versusHMECs. In a similar experiment, expression of SEQ ID NO:51 was decreasedat least two-fold in Sk-BR-3 cells versus HMECs. In a similarexperiment, expression of SEQ ID NO:54 was decreased at least two-foldin Sk-BR-3, T-47D, and MCF7 cells versus HMECs. In a similar experiment,expression of SEQ ID NO:57 was decreased at least two-fold in MDA-mb-231and MCF-10A cells versus HMECs. Therefore, in various embodiments, SEQID NO:51, SEQ ID NO:53-54, and SEQ ID NO:57 can be used for one or moreof the following: i) monitoring treatment of breast cancer, diagnosticassays for breast cancer, and iii) developing therapeutics and/or othertreatments for breast cancer.

In another example, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:53-54, and SEQID NO:57 were differentially expressed in breast carcinoma cell linesversus a cell line derived from a donor with non-malignant, fibrocysticbreast disease as determined by microarray analysis. Gene expressionprofiles of nonmalignant mammary epithelial cells were compared to geneexpression profiles of various breast carcinoma lines at differentstages of tumor progression. The cells were grown in defined serum-freeTCH medium, defined serum-free H14 medium, or the supplier's recommendedmedium to 70-80% confluence prior to RNA harvest and compared to MCF-10Acells grown in the same medium. Cell lines compared included: a)MCF-10A, a breast mammary gland (luminal ductal characteristics) cellline isolated from a 36-year-old woman with fibrocystic breast disease;b) MCF7, a nonmalignant breast adenocarcinoma cell line isolated fromthe pleural effusion of a 69-year-old female, c) T-47D, a breastcarcinoma cell line isolated from a pleural effusion obtained from a54-year-old female with an infiltrating ductal carcinoma of the breast,d) Sk-BR-3, a breast adenocarcinoma cell line isolated from a malignantpleural effusion of a 43-year-old female, e) BT-20, a breast carcinomacell line derived in vitro from the cells emigrating out of thin slicesof the tumor mass isolated from a 74-year-old female, f) MDA-mb-231, abreast tumor cell line isolated from the pleural effusion of a 51-yearold female, and g) MDA-nib-435S, a spindle shaped strain that evolvedfrom the parent line (435) isolated from the pleural effusion of a31-year-old female with metastatic, ductal adenocarcinoma of the breast.Expression of SEQ ID NO:45 was increased at least two-fold in MCF7 cellswhen grown in either the defined serum-free H14 medium or the supplier'srecommended medium as compared with MCF-10A cells grown under the sameconditions. In a similar experiment, expression of SEQ ID NO:51 wasdecreased at least two-fold in Sk-BR-3 cells when grown in any of thegrowth conditions as compared with MCF-10A cells grown under the sameconditions. In a similar experiment, expression of SEQ ID NO:53 wasincreased at least two-fold in MCF7 cells when grown in any of thegrowth conditions as compared with MCF-10A cells grown under the sameconditions. In a similar experiment, expression of SEQ ID NO:54 wasdecreased at least two-fold in Sk-BR-3 cells and T-47D cells when grownin any of the growth conditions as compared with MCF-10A cells grownunder the same conditions. In a similar experiment, expression of SEQ IDNO:57 was increased at least two-fold in MDA-mb-231 cells when grown ineither the defined serum-free H14 medium or the supplier's recommendedmedium as compared with MCF-10A cells grown under the same conditions.Therefore, in various embodiments, SEQ ID NO:45, SEQ ID NO:51, SEQ IDNO:53-54, and SEQ ID NO:57 can be used for one or more of the following:i) monitoring treatment of breast cancer, diagnostic assays for breastcancer, and developing therapeutics and/or other treatments for breastcancer.

In another example, expression of SEQ ID NO:47 was down-regulated in abreast cancer cell line (MCF7) treated with TNFα versus untreated MCF7cells as determined by microarray analysis. MCF7 cells were treated with10 ng/mL TNFα for 1, 4, 8, 12, 24, 48, and 72 hours. Treated cells werecompared to untreated cells kept in culture for the same amount of time.Expression of SEQ ID NO:47 was decreased at least two-fold in MCF7 cellstreated with 10 ng/mL TNFα for 4, 8, 24, or 48 hours as compared withuntreated MCF7 cells. Therefore, in various embodiments, SEQ ID NO:47can be used for one or more of the following: i) monitoring treatment ofbreast cancer, diagnostic assays for breast cancer, and developingtherapeutics and/or other treatments for breast cancer.

In another example, expression of SEQ ID NO:51 was down-regulated inovary tumor tissue versus normal ovary tissue as determined bymicroarray analysis. Expression of SEQ ID NO:51 was decreased at leasttwo-fold in ovary tumor tissue as compared with matched normal ovarytissue from the same donor in 1 of 2 donors tested. Therefore, invarious embodiments, SEQ ID NO:51 can be used for one or more of thefollowing: i) monitoring treatment of ovarian cancer, diagnostic assaysfor ovarian cancer, and developing therapeutics and/or other treatmentsfor ovarian cancer.

In another example, expression of SEQ ID NO:54 was down-regulated inbrain tissue from donors with Alzheimer's disease (AD) versus braintissue from a normal donor as determined by microarray analysis.Specific dissected brain regions from the cerebellum, dentate nucleus,and vermis of a normal donor were compared to: a) the correspondingregions dissected from the brain of a female with mild AD; and b) thecorresponding regions dissected from the brain of a female with severeAD. The diagnosis of normal or mild AD was established by a certifiedneuropathologist based on microscopic examination of multiple sectionsthroughout the brain. Expression of SEQ ID NO:54 was decreased at leasttwo-fold in the striatum and globus pallidus region of the brain of adonor with severe AD and a donor with mild AD as compared with thecorresponding region of the brain from a normal donor. Therefore, invarious embodiments, SEQ ID NO:54 can be used for one or more of thefollowing: i) monitoring treatment of AD, ii) diagnostic assays for AD,and iii) developing therapeutics and/or other treatments for AD.

In another example, expression of SEQ ID NO:57 was up-regulated in lungtumor tissue versus normal lung tissue as determined by microarrayanalysis. Expression of SEQ ID NO:57 was increased at least two-fold inlung tumor tissue as compared with matched normal lung tissue from thesame donor in 3 of 4 donors tested. Therefore, in various embodiments,SEQ ID NO:57 can be used for one or more of the following: i) monitoringtreatment of lung cancer, ii) diagnostic assays for lung cancer, andiii) developing therapeutics and/or other treatments for lung cancer.

In another example, expression of SEQ ID NO:57 was down-regulated to alesser extent in preadipocytes taken from an obese donor versuspreadipocytes taken from a non-obese donor as determined by microarrayanalysis. Primary subcutaneous preadipocytes were isolated from theadipose tissue of a non-obese donor, a 28-year-old healthy female withbody mass index (BMI) of 23.59, and an obese donor, a 40-year-oldhealthy female with a body mass index (BMI) of 32.47. The preadipocytesfrom each donor were cultured and induced to differentiate intoadipocytes by growing them in differentiation medium containing PPAR-γagonist and human insulin (Zen-Bio). Some thiazolidinediones or PPAR-γagonists, which bind and activate an orphan nuclear receptor, PPAR-γ,have been shown to induce human adipocyte differentiation. Thepreadipocytes were treated with human insulin and PPAR-γ agonist for 3days and subsequently were switched to medium containing insulin for arange of time periods ranging from one to 20 days before the cells werecollected for analysis. Differentiated adipocytes from each donor werecompared to untreated preadipocytes, maintained in culture in theabsence of differentiation-inducing agents, from the same donor. Between80% and 90% of the preadipocytes finally differentiated to adipocytes asobserved under phase contrast microscopy. Expression of SEQ ID NO:57 wasdecreased at least two-fold in differentiated preadipocytes from anormal donor versus non-differentiated preadipocytes from the samedonor. In contrast, no differential expression was seen indifferentiated preadipocytes from an obese donor versusnon-differentiated preadipocytes from the same donor. These data suggestthat SEQ ID NO:57 is differentially expressed in adipocytes from normalsubjects but not in adipocytes from obese subjects. Therefore, invarious embodiments, SEQ ID NO:57 can be used for one or more of thefollowing: i) monitoring treatment of diabetes mellitus and otherdisorders, such as obesity and hypertension ii) diagnostic assays fordiabetes mellitus and other disorders, such as obesity and hypertensioniii) developing therapeutics and/or other treatments for diabetesmellitus and other disorders, such as obesity and hypertension.

In another example, SEQ ID NO:47, SEQ ID NO:54, and SEQ ID NO:56 showedtissue-specific expression as determined by microarray analysis. RNAsamples isolated from a variety of normal human tissues were compared toa common reference sample. Tissues contributing to the reference samplewere selected for their ability to provide a complete distribution ofRNA in the human body and include brain (4%), heart (7%), kidney (3%),lung (8%), placenta (46%), small intestine (9%), spleen (3%), stomach(6%), testis (9%), and uterus (5%). The normal tissues assayed wereobtained from at least three different donors. RNA from each donor wasseparately isolated and individually hybridized to the microarray. Sincethese hybridization experiments were conducted using a common referencesample, differential expression values are directly comparable from onetissue to another. The expression of SEQ ID NO:47 was increased by atleast two-fold in small intestine and liver as compared to the referencesample. Therefore, SEQ ID NO:47 can be used as a tissue marker for smallintestine and liver. The expression of SEQ ID NO:54 was increased by atleast two-fold in brain (temporal cortex) and leukocytes as compared tothe reference sample. Therefore, SEQ ID NO:54 can be used as a tissuemarker for brain (temporal cortex) and leukocytes. The expression of SEQID NO:56 was increased by at least two-fold in brain as compared to thereference sample. Therefore, SEQ ID NO:56 can be used as a tissue markerfor brain.

In another example, SEQ ID NO:44 showed tissue-specific expression asdetermined by microarray analysis. RNA samples isolated from a varietyof normal human tissues were compared to a common reference sample.Tissues contributing to the reference sample were selected for theirability to provide a complete distribution of RNA in the human body andinclude brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%),small intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus(5%). The normal tissues assayed were obtained from at least threedifferent donors. RNA from each donor was separately isolated andindividually hybridized to the microarray. Since these hybridizationexperiments were conducted using a common reference sample, differentialexpression values are directly comparable from one tissue to another.The expression of SEQ ID NO:44 was increased by at least two-fold inleukocytes, thymus gland, and tonsil as compared to the referencesample. Therefore, SEQ ID NO:44 can be used as a tissue marker forleukocytes, thymus gland, and tonsil.

In another example, SEQ ID NO:48-50 showed tissue-specific expression asdetermined by microarray analysis. RNA samples isolated from a varietyof normal human tissues were compared to a common reference sample.Tissues contributing to the reference sample were selected for theirability to provide a complete distribution of RNA in the human body andinclude brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%),small intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus(5%). The normal tissues assayed were obtained from at least threedifferent donors. RNA from each donor was separately isolated andindividually hybridized to the microarray. Since these hybridizationexperiments were conducted using a common reference sample, differentialexpression values are directly comparable from one tissue to another.The expression of SEQ ID NO:48-50 was increased by at least two-fold inmuscle, adipose tissue, and liver as compared to the reference sample.Therefore, SEQ ID NO:48-50 can be used as a tissue marker for muscle,adipose tissue, and liver.

In another example, expression of SEQ ID NO:62 was up-regulated inbreast cancer cell lines versus a breast epithelial cell line derivedfrom normal breast tissue as determined by microarray analysis. Geneexpression profiles of nonmalignant mammary epithelial cells werecompared to gene expression profiles of various breast carcinoma linesat different stages of tumor progression. The cells were grown indefined serum-free H14 medium to 70-80% confluence prior to RNA harvest.Cell lines compared included: a) HMEC, a primary breast epithelial cellline isolated from a normal donor, b) MCF-10A, a breast mammary glandcell line isolated from a 36-year-old woman with fibrocystic breastdisease, c) MCF7, a nonmalignant breast adenocarcinoma cell lineisolated from the pleural effusion of a 69-year-old female, d) T-47D, abreast carcinoma cell line isolated from a pleural effusion obtainedfrom a 54-year-old female with an infiltrating ductal carcinoma of thebreast, e) Sk-BR-3, a breast adenocarcinoma cell line isolated from amalignant pleural effusion of a 43-year-old female, f) BT-20, a breastcarcinoma cell line derived in vitro from cells emigrating out of thinslices of the tumor mass isolated from a 74-year-old female, g)MDA-mb-231, a breast tumor cell line isolated from the pleural effusionof a 51-year-old female, and h) MDA-mb-435S, a spindle-shaped strainthat evolved from the parent line (435) isolated by R. Cailleau frompleural effusion of a 31-year-old female with metastatic, ductaladenocarcinoma of the breast. Expression of SEQ ID NO:62 was increasedat least two-fold in two (MDA-mb-231 and MCF-10A) of seven breast cancercell lines tested compared to HMECs. Therefore, in various embodiments,SEQ ID NO:62 can be used for one or more of the following: i) monitoringtreatment of breast cancer, ii) diagnostic assays for breast cancer, andiii) developing therapeutics and/or other treatments for breast cancer.

In another example, expression of SEQ ID NO:62 was up-regulated in lungcancer tissue versus normal lung tissue as determined by microarrayanalysis. Expression of SEQ ID NO:62 was increased at least two-fold inlung tumor tissue versus matched normal lung tissue from the same donorin three of three donors with squamous cell cancer tested. Therefore, invarious embodiments, SEQ ID NO:62 can be used for one or more of thefollowing: i) monitoring treatment of lung cancer, ii) diagnostic assaysfor lung cancer, and iii) developing therapeutics and/or othertreatments for lung cancer.

In another example, expression of SEQ ID NO:62 was down-regulated to alesser extent in preadipocytes taken from an obese donor versuspreadipocytes taken from a non-obese donor as determined by microarrayanalysis. Primary subcutaneous preadipocytes were isolated from theadipose tissue of a non-obese donor, a 28-year-old healthy female withbody mass index (BMI) of 23.59, and an obese donor, a 40-year-oldhealthy female with a body mass index (BMI) of 32.47. The preadipocytesfrom each donor were cultured and induced to differentiate intoadipocytes by growing them in differentiation medium containing PPAR-γagonist and human insulin (Zen-Bio). Some thiazolidinediones or PPAR-γagonists, which bind and activate an orphan nuclear receptor, PPAR-γ,have been shown to induce human adipocyte differentiation. Thepreadipocytes were treated with human insulin and PPAR-γ agonist for 3days and subsequently were switched to medium containing insulin for arange of time periods ranging from one to 20 days before the cells werecollected for analysis. Differentiated adipocytes from each donor werecompared to untreated preadipocytes, maintained in culture in theabsence of differentiation-inducing agents, from the same donor. Between80% and 90% of the preadipocytes finally differentiated to adipocytes asobserved under phase contrast microscopy. Expression of SEQ ID NO:62 wasdecreased at least two-fold in differentiated preadipocytes from anormal donor versus non-differentiated preadipocytes from the samedonor. In contrast, no differential expression was seen indifferentiated preadipocytes from an obese donor versusnon-differentiated preadipocytes from the same donor. These data suggestthat SEQ ID NO:62 is differentially expressed in adipocytes from normalsubjects but not in adipocytes from obese subjects. Therefore, invarious embodiments, SEQ ID NO:62 can be used for one or more of thefollowing: i) monitoring treatment of diabetes mellitus and otherdisorders, such as obesity and hypertension ii) diagnostic assays fordiabetes mellitus and other disorders, such as obesity and hypertensioniii) developing therapeutics and/or other treatments for diabetesmellitus and other disorders, such as obesity and hypertension.

In another example, expression of SEQ ID NO:69 was down-regulated indiseased lung tissue versus normal lung tissue as determined bymicroarray analysis. Expression of SEQ ID NO:69 was decreased at leasttwo-fold in the lung tumor tissue with squamous cell carcinoma ascompared to grossly uninvolved lung tissue from the same donor using apair comparison experimental design. Therefore, in various embodiments,SEQ ID NO:69 can be used for one or more of the following: i) monitoringtreatment of lung cancer, diagnostic assays for lung cancer, anddeveloping therapeutics and/or other treatments for lung cancer.

In another example, expression of SEQ ID NO:74 was downregulated inbrain tissue affected by Alzheimer's Disease versus normal brain tissueas determined by microarray analysis. Specific dissected brain regionsfrom the brain patients with AD were compared to dissected regions fromnormal brain. The diagnosis of normal or AD was established by acertified neuropathologist based on microscopic examination of multiplesections throughout the brain. Expression of SEQ ID NO:74 was decreasedat least two-fold in 7 of 10 AD-affected tissue samples. Therefore, invarious embodiments, SEQ ID NO:74 can be used for one or more of thefollowing: i) monitoring treatment of Alzheimer's Disease, ii)diagnostic assays for Alzheimer's Disease, and iii) developingtherapeutics and/or other treatments for Alzheimer's Disease asdetermined by microarray analysis.

As another example, SEQ ID NO:72 and SEQ ID NO:74 were downregulated inbreast cancer cells versus nonmalignant mammary epithelial cells, asdetermined by microarray analysis. Cell lines compared included: a)MCF-10A, a breast mammary gland (luminal ductal characteristics) cellline isolated from a 36-year-old woman with fibrocystic breast disease,b) MCF7, a nonmalignant breast adenocarcinoma cell line isolated fromthe pleural effusion of a 69-year-old female, c) BT-20, a breastcarcinoma cell line derived in vitro from the cells emigrating out ofthin slices of tumor mass isolated from a 74-year-old female, d) T-47D,a breast carcinoma cell line isolated from a pleural effusion obtainedfrom a 54-year-old female with an infiltrating ductal carcinoma of thebreast, e) Sk-BR-3, a breast adenocarcinoma cell line isolated from amalignant pleural effusion of a 43-year-old female, f) MDA-mb-231, abreast tumor cell line isolated from the pleural effusion of a51-year-old female, g) MDA-mb-435S, a spindle-shaped strain that evolvedfrom the parent line (435) isolated by R. Cailleau from pleural effusionof a 31-year-old female with metastatic, ductal adenocarcinoma of thebreast, and h) HMEC, a primary breast epithelial cell line isolated froma normal donor. Expression of SEQ ID NO:72 was decreased at leasttwo-fold in the Sk-BR-3, BT-20, MDA-mb-435S, T-47D, and MCF7 cell linesas compared to the normal breast epithelial cells. Expression of SEQ IDNO:74 was decreased at least two-fold in the MCF-10A, T-47D, Sk-BR-3,and MCF7 cell lines as compared to the normal breast epithelial cells.Therefore, in various embodiments, SEQ ID NO:72 and SEQ ID NO:74 can beused for one or more of the following: i) monitoring treatment of breastcancer, diagnostic assays for breast cancer, and developing therapeuticsand/or other treatments for breast cancer as determined by microarrayanalysis.

As another example, SEQ ID NO:74 and SEQ ID NO:77 showed tissue-specificexpression as determined by microarray analysis. RNA samples isolatedfrom a variety of normal human tissues were compared to a commonreference sample. Tissues contributing to the reference sample wereselected for their ability to provide a complete distribution of RNA inthe human body and include brain (4%), heart (7%), kidney (3%), lung(8%), placenta (46%), small intestine (9%), spleen (3%), stomach (6%),testis (9%), and uterus (5%). The normal tissues assayed were obtainedfrom at least three different donors. RNA from each donor was separatelyisolated and individually hybridized to the microarray. Since thesehybridization experiments were conducted using a common referencesample, differential expression values are directly comparable from onetissue to another. The expression of SEQ ID NO:74 was increased by atleast two-fold in brain cortex tissue as compared to the referencesample. Therefore, SEQ ID NO:74 can be used as a tissue marker for braincortex tissue. The expression of SEQ ID NO:77 was increased by at leasttwo-fold in heart tissue as compared to the reference sample. Therefore,SEQ ID NO:77 can be used as a tissue marker for heart tissue.

XII. Complementary Polynucleotides

Sequences complementary to the KPP-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring KPP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of KPP. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the KPP-encoding transcript.

XIII. Expression of KPP

Expression and purification of KPP is achieved using bacterial orvirus-based expression systems. For expression of KPP in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express KPP uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof KPP in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known, as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding KPP by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus (Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.Gene Ther. 7:1937-1945).

In most expression systems, KPP is synthesized as a fusion protein with,e.g., glutathione S-transferase (GST) or a peptide epitope tag, such asFLAG or 6-His SEQ ID NO: 87), permitting rapid, single-step,affinity-based purification of recombinant fusion protein from crudecell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum,enables the purification of fusion proteins on immobilized glutathioneunder conditions that maintain protein activity and antigenicity(Amersham Biosciences). Following purification, the GST moiety can beproteolytically cleaved from KPP at specifically engineered sites. FLAG,an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His (SEQ ID NO: 87), a stretch of six consecutivehistidine residues, enables purification on metal-chelate resins(QIAGEN). Methods for protein expression and purification are discussedin Ausubel et al. (supra, ch. 10 and 16). Purified KPP obtained by thesemethods can be used directly in the assays shown in Examples XVII,XVIII, XIX, XX, and XXI, where applicable.

XIV. Functional Assays

KPP function is assessed by expressing the sequences encoding KPP atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude PCMV SPORT plasmid (Invitrogen, Carlsbad Calif.) and PCR3.1plasmid (Invitrogen), both of which contain the cytomegaloviruspromoter. 5-10 μg of recombinant vector are transiently transfected intoa human cell line, for example, an endothelial or hematopoietic cellline, using either liposome formulations or electroporation. 1-2 μg ofan additional plasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;BD Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM),an automated, laser optics-based technique, is used to identifytransfected cells expressing GFP or CD64-GFP and to evaluate theapoptotic state of the cells and other cellular properties. FCM detectsand quantifies the uptake of fluorescent molecules that diagnose eventspreceding or coincident with cell death. These events include changes innuclear DNA content as measured by staining of DNA with propidiumiodide; changes in cell size and granularity as measured by forwardlight scatter and 90 degree side light scatter; down-regulation of DNAsynthesis as measured by decrease in bromodeoxyuridine uptake;alterations in expression of cell surface and intracellular proteins asmeasured by reactivity with specific antibodies; and alterations inplasma membrane composition as measured by the binding offluorescein-conjugated Annexin V protein to the cell surface. Methods inflow cytometry are discussed in Ormerod, M. G. (1994; Flow Cytometry,Oxford, New York N.Y.).

The influence of KPP on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding KPPand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding KPP and other genes of interest canbe analyzed by northern analysis or microarray techniques.

XV. Production of KPP Specific Antibodies

KPP substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizeanimals (e.g., rabbits, mice, etc.) and to produce antibodies usingstandard protocols.

Alternatively, the KPP amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art (Ausubel et al.,supra, ch. 11).

Typically, oligopeptides of about 15 residues in length are synthesizedusing an ABI 431A peptide synthesizer (Applied Biosystems) using FMOCchemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity (Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide and anti-KPP activity by, forexample, binding the peptide or KPP to a substrate, blocking with 1%BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG.

XVI. Purification of Naturally Occurring KPP Using Specific Antibodies

Naturally occurring or recombinant KPP is substantially purified byimmunoaffinity chromatography using antibodies specific for KPP. Animmunoaffinity column is constructed by covalently coupling anti-KPPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Biosciences). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing KPP are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof KPP (e.g., high ionic strength buffers in the presence of detergent).The column is eluted under conditions that disrupt antibody/KPP binding(e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope,such as urea or thiocyanate ion), and KPP is collected.

XVII. Identification of Molecules Which Interact with KPP

KPP, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973) Biochem. J.133:529-539). Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled KPP, washed, and anywells with labeled KPP complex are assayed. Data obtained usingdifferent concentrations of KPP are used to calculate values for thenumber, affinity, and association of KPP with the candidate molecules.

Alternatively, molecules interacting with KPP are analyzed using theyeast two-hybrid system as described in Fields, S, and O, Song (1989;Nature 340:245-246), or using commercially available kits based on thetwo-hybrid system, such as the MATCHMAKER system (BD Clontech).

KPP may also be used in the PATHCALLING process (CuraGen Corp., NewHaven Conn.) which employs the yeast two-hybrid system in ahigh-throughput manner to determine all interactions between theproteins encoded by two large libraries of genes (Nandabalan, K. et al.(2000) U.S. Pat. No. 6,057,101).

XVIII. Demonstration of KPP Activity

Generally, protein kinase activity is measured by quantifying thephosphorylation of a protein substrate by KPP in the presence of[γ-³²P]ATP. KPP is incubated with the protein substrate, ³²P-ATP, and anappropriate kinase buffer. The ³²P incorporated into the substrate isseparated from free ³²P-ATP by electrophoresis and the incorporated ³²Pis counted using a radioisotope counter. The amount of incorporated ³²Pis proportional to the activity of KPP. A determination of the specificamino acid residue phosphorylated is made by phosphoamino acid analysisof the hydrolyzed protein.

In one alternative, protein kinase activity is measured by quantifyingthe transfer of gamma phosphate from adenosine triphosphate (ATP) to aserine, threonine or tyrosine residue in a protein substrate. Thereaction occurs between a protein kinase sample with a biotinylatedpeptide substrate and gamma ³²P-ATP. Following the reaction, free avidinin solution is added for binding to the biotinylated ³²P-peptideproduct. The binding sample then undergoes a centrifugal ultrafiltrationprocess with a membrane which will retain the product-avidin complex andallow passage of free gamma ³²P-ATP. The reservoir of the centrifugedunit containing the ³²P-peptide product as retentate is then counted ina scintillation counter. This procedure allows the assay of any type ofprotein kinase sample, depending on the peptide substrate and kinasereaction buffer selected. This assay is provided in kit form (ASUA,Affinity Ultrafiltration Separation Assay, Transbio Corporation,Baltimore Md., U.S. Pat. No. 5,869,275). Suggested substrates and theirrespective enzymes include but are not limited to: Histone H1 (Sigma)and p34^(cdc2)kinase, Annexin I, Angiotensin (Sigma) and EGF receptorkinase, Annexin II and src kinase, ERK1 & ERK2 substrates and MEK, andmyelin basic protein and ERK (Pearson, J. D. et al. (1991) MethodsEnzymol. 200:62-81).

In another alternative, protein kinase activity of KPP is demonstratedin an assay containing KPP, 50 μl of kinase buffer, 1 μg substrate, suchas myelin basic protein (MBP) or synthetic peptide substrates, 1 mM DTT,10 μg ATP, and 0.5 μCi [γ-³²P]ATP. The reaction is incubated at 30° C.for 30 minutes and stopped by pipetting onto P81 paper. Theunincorporated [γ-³²P]ATP is removed by washing and the incorporatedradioactivity is measured using a scintillation counter. Alternatively,the reaction is stopped by heating to 100° C. in the presence of SDSloading buffer and resolved on a 12% SDS polyacrylamide gel followed byautoradiography. The amount of incorporated ³²P is proportional to theactivity of KPP.

In yet another alternative, adenylate kinase or guanylate kinaseactivity of KPP may be measured by the incorporation of ³²P from[γ-³²P]ATP into ADP or GDP using a gamma radioisotope counter. KPP, in akinase buffer, is incubated together with the appropriate nucleotidemono-phosphate substrate (AMP or GMP) and ³²P-labeled ATP as thephosphate donor. The reaction is incubated at 37° C. and terminated byaddition of trichloroacetic acid. The acid extract is neutralized andsubjected to gel electrophoresis to separate the mono-, di-, andtriphosphonucleotide fractions. The diphosphonucleotide fraction isexcised and counted. The radioactivity recovered is proportional to theactivity of KPP.

In yet another alternative, other assays for KPP include scintillationproximity assays (SPA), scintillation plate technology and filterbinding assays. Useful substrates include recombinant proteins taggedwith glutathione transferase, or synthetic peptide substrates taggedwith biotin.

Inhibitors of KPP activity, such as small organic molecules, proteins orpeptides, may be identified by such assays.

In another alternative, phosphatase activity of KPP is measured by thehydrolysis of para-nitrophenyl phosphate (PNPP). KPP is incubatedtogether with PNPP in HEPES buffer pH 7.5, in the presence of 0.1%β-mercaptoethanol at 37° C. for 60 min. The reaction is stopped by theaddition of 6 ml of 10 N NaOH (Diamond, R. H. et al. (1994) Mol. Cell.Biol. 14:3752-62). Alternatively, acid phosphatase activity of KPP isdemonstrated by incubating KPP-containing extract with 100 μl of 10 mMPNPP in 0.1 M sodium citrate, pH 4.5, and 50 μl of 40 mM NaCl at 37° C.for 20 min. The reaction is stopped by the addition of 0.5 ml of 0.4 Mglycine/NaOH, pH 10.4 (Saftig, P. et al. (1997) J. Biol. Chem.272:18628-18635). The increase in light absorbance at 410 tun resultingfrom the hydrolysis of PNPP is measured using a spectrophotometer. Theincrease in light absorbance is proportional to the activity of KPP inthe assay.

In the alternative, KPP activity is determined by measuring the amountof phosphate removed from a phosphorylated protein substrate. Reactionsare performed with 2 or 4 nM KPP in a final volume of 30 μl containing60 mM Tris, pH 7.6, 1 mM EDTA, 1 mM EGTA, 0.1% β-mercaptoethanol and 10μM substrate, ³²P-labeled on serine/threonine or tyrosine, asappropriate. Reactions are initiated with substrate and incubated at 30°C. for 10-15 min. Reactions are quenched with 450 μl of 4% (w/v)activated charcoal in 0.6 M HCl, 90 mM Na₄P₂O₇, and 2 mM NaH₂PO₄, thencentrifuged at 12,000×g for 5 min. Acid-soluble ³²Pi is quantified byliquid scintillation counting (Sinclair, C. et al. (1999) J. Biol. Chem.274:23666-23672).

XIX. Kinase Binding Assay

Binding of KPP to a FLAG-CD44 cyt fusion protein can be determined byincubating KPP with anti-KPP-conjugated immunoaffinity beads followed byincubating portions of the beads (having 10-20 ng of protein) with 0.5ml of a binding buffer (20 mM Tris-HCL (pH 7.4), 150 mM NaCl, 0.1%bovine serum albumin, and 0.05% Triton X-100) in the presence of¹²⁵I-labeled FLAG-CD44cyt fusion protein (5,000 cpm/ng protein) at 4° C.for 5 hours. Following binding, beads were washed thoroughly in thebinding buffer and the bead bound radioactivity measured in ascintillation counter (Bourguignon, L. Y. W. et al. (2001) J. Biol.Chem. 276:7327-7336). The amount of incorporated ³²P is proportional tothe amount of bound KPP.

XX. Identification of KPP Inhibitors

Compounds to be tested are arrayed in the wells of a 384-well plate invarying concentrations along with an appropriate buffer and substrate,as described in the assays in Example XVII. KPP activity is measured foreach well and the ability of each compound to inhibit KPP activity canbe determined, as well as the dose-response kinetics. This assay couldalso be used to identify molecules which enhance KPP activity.

XXI. Identification of KPP Substrates

A KPP “substrate-trapping” assay takes advantage of the increasedsubstrate affinity that may be conferred by certain mutations in the PTPsignature sequence of protein tyrosine phosphatases. KPP bearing thesemutations form a stable complex with their substrate; this complex maybe isolated biochemically. Site-directed mutagenesis of invariantresidues in the PTP signature sequence in a clone encoding the catalyticdomain of KPP is performed using a method standard in the art or acommercial kit, such as the MUM-GENE kit from BIO-RAD. For expression ofKPP mutants in Escherichia coli, DNA fragments containing the mutationare exchanged with the corresponding wild-type sequence in an expressionvector bearing the sequence encoding KPP or a glutathione S-transferase(GST)-KPP fusion protein. KPP mutants are expressed in E. coli andpurified by chromatography.

The expression vector is transfected into COS1 or 293 cells via calciumphosphate-mediated transfection with 20 μg of CsCl-purified DNA per10-cm dish of cells or 8 μg per 6-cm dish. Forty-eight hours aftertransfection, cells are stimulated with 100 ng/ml epidermal growthfactor to increase tyrosine phosphorylation in cells, as the tyrosinekinase EGFR is abundant in COS cells. Cells are lysed in 50 mM Tris.HCl,pH 7.5/5 mM EDTA/150 mM NaCl/1% Triton X-100/5 mM iodoacetic acid/10 mMsodium phosphate/10 mM NaF/5 μg/ml leupeptin/5 μg/ml aprotinin/1 mMbenzamidine (1 nil per 10-cm dish, 0.5 ml per 6-cm dish). KPP isimmunoprecipitated from lysates with an appropriate antibody. GST-KPPfusion proteins are precipitated with glutathione-Sepharose, 4 μg of mAbor 10 μl of beads respectively per mg of cell lysate. Complexes can bevisualized by PAGE or further purified to identify substrate molecules(Flint, A. J. et al. (1997) Proc. Natl. Acad. Sci. USA 94:1680-1685).

Various modifications and variations of the described compositions,methods, and systems of the invention will be apparent to those skilledin the art without departing from the scope and spirit of the invention.It will be appreciated that the invention provides novel and usefulproteins, and their encoding polynucleotides, which can be used in thedrug discovery process, as well as methods for using these compositionsfor the detection, diagnosis, and treatment of diseases and conditions.Although the invention has been described in connection with certainembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Nor shouldthe description of such embodiments be considered exhaustive or limitthe invention to the precise forms disclosed. Furthermore, elements fromone embodiment can be readily recombined with elements from one or moreother embodiments. Such combinations can form a number of embodimentswithin the scope of the invention. It is intended that the scope of theinvention be defined by the following claims and their equivalents.

TABLE 1 Polypeptide Incyte Polynucleotide Incyte Incyte Project ID SEQID NO: Polypeptide ID SEQ ID NO: Polynucleotide ID Incyte Full LengthClones 7517831 1 7517831CD1 44 7517831CB1 90040615CA2 7520272 27520272CD1 45 7520272CB1 95114642CA2, 95114758CA2, 95162524CA2,95162564CA2 7521279 3 7521279CD1 46 7521279CB1 95121315CA2, 95121539CA27523965 4 7523965CD1 47 7523965CB1 95141143CA2 7524016 5 7524016CD1 487524016CB1 95183446CA2 7524680 6 7524680CD1 49 7524680CB1 7524757 77524757CD1 50 7524757CB1 7516229 8 7516229CD1 51 7516229CB1 7516525 97516525CD1 52 7516525CB1 7516533 10 7516533CD1 53 7516533CB1 90041659CA27516613 11 7516613CD1 54 7516613CB1 90136641CA2 7517068 12 7517068CD1 557517068CB1 7517148 13 7517148CD1 56 7517148CB1 7517238 14 7517238CD1 577517238CB1 90094269CA2 7518685 15 7518685CD1 58 7518685CB1 7520192 167520192CD1 59 7520192CB1 90111670CA2 7520428 17 7520428CD1 60 7520428CB190198192CA2 7522586 18 7522586CD1 61 7522586CB1 7524017 19 7524017CD1 627524017CB1 7525773 20 7525773CD1 63 7525773CB1 7525861 21 7525861CD1 647525861CB1 95132479CA2, 95206437CA2, 95206561CA2 2509577 22 2509577CD165 2509577CB1 7505222 23 7505222CD1 66 7505222CB1 7524408 24 7524408CD167 7524408CB1 7526163 25 7526163CD1 68 7526163CB1 7526158 26 7526158CD169 7526158CB1 7519807 27 7519807CD1 70 7519807CB1 90124401CA2 7526180 287526180CD1 71 7526180CB1 7526185 29 7526185CD1 72 7526185CB1 7526192 307526192CD1 73 7526192CB1 7526193 31 7526193CD1 74 7526193CB1 7526196 327526196CD1 75 7526196CB1 7526198 33 7526198CD1 76 7526198CB1 7526208 347526208CD1 77 7526208CB1 7526212 35 7526212CD1 78 7526212CB1 7526213 367526213CD1 79 7526213CB1 7526214 37 7526214CD1 80 7526214CB1 7526228 387526228CD1 81 7526228CB1 7526246 39 7526246CD1 82 7526246CB1 7526258 407526258CD1 83 7526258CB1 7526311 41 7526311CD1 84 7526311CB1 7526315 427526315CD1 85 7526315CB1 7526442 43 7526442CD1 86 7526442CB1

TABLE 2 Genbank Polypeptide Incyte ID NO: or SEQ Polypeptide PROTEOMEProbability ID NO: ID ID NO: Score Annotation 1 7517831CD1 g7752084.3E−21 [Homo sapiens] p56lck Vogel, L. B. et al., p70 phosphorylationand binding to p56lck is an early event in interleukin-2-induced onsetof cell cycle progression in T-lymphocytes, J. Biol. Chem. 270,2506-2511 (1995) 342146|LCK 7.2E−20 [Homo sapiens][Proteinkinase;Transferase] Lymphocyte-specific protein tyrosine kinase,tyrosine kinase that is involved in T cell receptor signaling throughRas and MAPK pathways, activator of CASP8 in radiation-inducedapoptosis; gene defect correlates with immunodeficiency plus CD4lymphopenia Su, S. B. et al., Inhibition of tyrosine kinase activationblocks the down-regulation of CXC chemokine receptor 4 by HIV-1 gp120 inCD4+ T cells, J Immunol 162, 7128-32 (1999). 780711|Lck 8.0E−17 [Musmusculus][Protein kinase;Transferase] Lymphocyte-specific proteintyrosine kinase, tyrosine kinase that is involved in T cell receptorsignaling through Ras and MAPK pathways, regulates T cell developmentand apoptosis; human gene defect correlates with immunodeficiency plusCD4 lymphopenia Legname, G. et al., Inducible expression of a p56Lcktransgene reveals a central role for Lck in the differentiation of CD4SP thymocytes, Immunity 12, 537-46 (2000). 2 7520272CD1 g439226 4.0E−152 [Homo sapiens] fructose-1,6-bisphosphatase Kikawa, Y. et al.,cDNA sequences encoding human fructose 1,6-bisphosphatase frommonocytes, liver and kidney: Application of monocytes to molecularanalysis of human fructose 1,6-bisphosphatase deficiency, Cell. Mol.Biol. Res. 199, 687-693 (1994) 753731|FBP1  3.0E−153 [Homosapiens][Other phosphatase;Hydrolase] Fructose-1,6-bisphosphatase 1(liver), hydrolyzes fructose-1,6-bisphosphate to fructose-6-phosphateand inorganic phosphate, regulatory step in gluconeogenesis; deficiencyis associated with metabolic acidosis and fasting hypoglycemiael-Maghrabi, M. R. et al., Isolation of a human liverfructose-1,6-bisphosphatase cDNA and expression of the protein inEscherichia coli. Role of ASP-118 and ASP-121 in catalysis, J Biol Chem268, 9466-72 (1993). 3 7521279CD1 g1905761  4.1E−233 [Homo sapiens]6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase Sakai, A. et al.,Cloning of cDNA encoding for a novel isozyme of fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase from human placenta, J. Biochem. 119,506-511 (1996) 341042|PFKFB4  3.0E−234 [Homo sapiens][Transferase;Otherkinase;Other phosphatase;Hydrolase]6-phosphofructo-2- kinase,fructose-2,6-biphosphatase, testis form, synthesizes and degradesfructose-2,6- bisphosphate and may be involved in the regulation ofglycolysis Manzano, A. et al., Cloning, expression and chromosomallocalization of a human testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene, Gene 229, 83-9(1999). 609815|Pfkfb4  1.1E−227 [Rattus norvegicus][Transferase;Otherkinase;Other phosphatase;Hydrolase] 6- phosphofructo-2-kinase,fructose-2,6-biphosphatase, testis form, synthesizes and degradesfructose-2,6-bisphosphate and may be involved in the regulation ofglycolysis Li, L. et al., Expression of chicken liver6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase in Escherichiacoli, Biochem Biophys Res Commun 209, 883-93 (1995). 4 7523965CD1g2661752  1.1E−120 [Homo sapiens] phosphoenolpyruvate carboxykinase(GTP) Modaressi, S. et al., Human mitochondrial phosphoenolpyruvatecarboxykinase 2 gene. Structure, chromosomal localization andtissue-specific expression, Biochem. J. 333 (Pt 2), 359-366 (1998)341026|PCK2  5.7E−121 [Homo sapiens][Lyase;Otherkinase][Cytoplasmic;Mitochondrial] Phosphoenolpyruvate carboxykinase 2,catalyzes the formation of phosphoenolpyruvate by decarboxylation ofoxaloacetate, rate-limiting step of gluconeogenesis Modaressi, S. etal., Molecular cloning, sequencing and expression of the cDNA of themitochondrial form of phosphoenolpyruvate carboxykinase from humanliver, Biochem J 315, 807-14 (1996). 586739|Pck1 2.6E−68 [Musmusculus][Lyase;Other kinase][Cytoplasmic] Cytosolic phosphoenolpyruvatecarboxykinase, catalyzes the formation of phosphoenolpyruvate bydecarboxylation of oxaloacetate She, P. et al., Phosphoenolpyruvatecarboxykinase is necessary for the integration of hepatic energymetabolism, Mol Cell Biol 20, 6508-17 (2000). 5 7524016CD1 g355031.7E−94 [Homo sapiens] 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (AA 1-471) Lange, A. J. et al., Sequence of human liver6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, Nucleic AcidsRes. 18, 3652 (1990) 336898|PFKFB1 1.2E−95 [Homo sapiens][Proteinphosphatase;Transferase;Other phosphatase;Other kinase;Hydrolase]6-phosphofructo-2-kinase, fructose-2,6-biphosphatase 1, liver and muscleform, enzyme involved in regulating glycolysis, catalyzes the synthesisand degradation of fructose-2,6-bisphosphate Lange, A. J. et al.,Expression of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli. Role of N-2 proline in degradationof the protein, J Biol Chem 268, 8078-84 (1993). 430618|Pfkfb1 9.3E−89[Rattus norvegicus][Protein phosphatase;Transferase;Otherphosphatase;Other kinase;Hydrolase] 6-phosphofructo-2-kinase,fructose-2,6-biphosphatase 1, liver and muscle form, enzyme involved inregulating glycolysis, catalyzes the synthesis and degradation offructose-2,6-bisphosphate Kurland, I. J. et al., Rat liver6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties ofphospho- and dephospho- forms and of two mutants in which Ser32 has beenchanged by site-directed mutagenesis, J Biol Chem 267, 4416-23 (1992). 67524680CD1 g35503  1.3E−215 [Homo sapiens]6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase (AA 1-471) Lange,A. J. et al., Sequence of human liver6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, Nucleic AcidsRes. 18, 3652 (1990) 336898|PFKFB1  9.1E−217 [Homo sapiens][Proteinphosphatase;Transferase;Other phosphatase;Other kinase;Hydrolase]6-phosphofructo-2-kinase, fructose-2,6-biphosphatase 1, liver and muscleform, enzyme involved in regulating glycolysis, catalyzes the synthesisand degradation of fructose-2,6-bisphosphate Lange, A. J. et al.,Expression of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli. Role of N-2 proline in degradationof the protein, J Biol Chem 268, 8078-84 (1993). 430618|Pfkfb1  1.2E−207[Rattus norvegicus][Protein phosphatase;Transferase;Otherphosphatase;Other kinase;Hydrolase] 6-phosphofructo-2-kinase,fructose-2,6-biphosphatase 1, liver and muscle form, enzyme involved inregulating glycolysis, catalyzes the synthesis and degradation offructose-2,6-bisphosphate Kurland, I. J. et al., Rat liver6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties ofphospho- and dephospho- forms and of two mutants in which Ser32 has beenchanged by site-directed mutagenesis, J Biol Chem 267, 4416-23 (1992). 77524757CD1 g35503  3.7E−223 [Homo sapiens]6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase (AA 1-471) Lange,A. J. et al., Sequence of human liver6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, Nucleic AcidsRes. 18, 3652 (1990) 336898|PFKFB1  2.7E−224 [Homo sapiens][Proteinphosphatase;Transferase;Other phosphatase;Other kinase;Hydrolase]6-phosphofructo-2-kinase, fructose-2,6-biphosphatase 1, liver and muscleform, enzyme involved in regulating glycolysis, catalyzes the synthesisand degradation of fructose-2,6-bisphosphate Lange, A. J. et al.,Expression of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli. Role of N-2 proline in degradationof the protein, J Biol Chem 268, 8078-84 (1993). 430618|Pfkfb1  3.7E−216[Rattus norvegicus][Protein phosphatase;Transferase;Otherphosphatase;Other kinase;Hydrolase] 6-phosphofructo-2-kinase,fructose-2,6-biphosphatase 1, liver and muscle form, enzyme involved inregulating glycolysis, catalyzes the synthesis and degradation offructose-2,6-bisphosphate Kurland, I. J. et al., Rat liver6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties ofphospho- and dephospho- forms and of two mutants in which Ser32 has beenchanged by site-directed mutagenesis, J Biol Chem 267, 4416-23 (1992). 87516229CD1 g6760472  3.0E−190 [Homo sapiens] type IIphosphatidy]inositol-4-phosphate 5-kinase 53K isoform Boronenkov, I. V.et al., The sequence of phosphatidylinositol-4-phosphate 5-kinasedefines a novel family of lipid kinases, J. Biol. Chem. 270, 2881-2884(1995) 568490|  2.1E−191 [Homo sapiens][Transferase;Other kinase]Phosphatidylinositol-4-phosphate 5-kinase type PIP5K2A II, alpha, amember of a family of kinases responsible for the synthesis ofPtdIns(4,5)P2 Boronenkov, I. V. et al., The sequence ofphosphatidylinositol-4-phosphate 5-kinase defines a novel family oflipid kinases, J Biol Chem 270, 2881-4 (1995). 757680|Pip5k2a  5.0E−188[Rattus norvegicus] Phosphatidylinositol-4-phosphate 5-kinase type IIalpha Itoh, T. et al., Autophosphorylation of type Iphosphatidylinositol phosphate kinase regulates its lipid kinaseactivity, J Biol Chem 275, 19389-94 (2000). 9 7516525CD1 g23499314 6.7E−270 [Homo sapiens] (AF425232) CaMKK alpha protein 716531| 4.6E−271 [Homo sapiens] Protein with strong similarity tocalcium-calmodulin-dependent protein DKFZp7 kinase kinase 1 alpha (ratCamkk1), which phosphorylates and activates Ca(2+)-calmodulin 61M0423(CaM)-dependent kinase I and IV but not CaM kinase II, contains aprotein kinase domain 711580|  2.4E−254 [Rattus norvegicus][Proteinkinase;Transferase] Calcium-calmodulin-dependent protein Camkk1 kinasekinase 1 alpha, phosphorylates and activates Ca(2+)-calmodulin(CaM)-dependent kinase I and IV but not CaM kinase II, involved inCa(2+)-calmodulin signaling Okuno, S. et al., Regulation ofCa(2+)/Calmodulin-Dependent Protein Kinase Kinase alpha bycAMP-Dependent Protein Kinase: I. Biochemical Analysis, J Biochem(Tokyo) 130, 503-13. (2001). 10 7516533CD1 g189508  3.6E−240 [Homosapiens] p70 ribosomal S6 kinase alpha-I Grove, J. R. et al., Cloningand expression of two human p70 S6 kinase polypeptides differing only attheir amino termini, Mol. Cell. Biol. 11, 5541-5550 (1991) 337822| 2.5E−241 [Homo sapiens][Protein kinase;Transferase] Ribosomal proteinS6 kinase, 70 kD, a member RPS6KB1 of the ribosomal protein S6 kinase(RSK) family of protein kinases, insulin and mitogen activated, andplays roles in cell cycle progression and control of cell proliferationBrenneisen, P. et al., Activation of p70 ribosomal protein S6 kinase isan essential step in the DNA damage-dependent signaling pathwayresponsible for the ultraviolet B-mediated increase in interstitialcollagenase (MMP-1) and stromelysin-1 (MMP-3) protein levels in humandermal fibroblasts, J Biol Chem 275, 4336-44. (2000). 711952|Rps6kb1 5.3E−239 [Rattus norvegicus][Protein kinase;Transferase] Ribosomalprotein S6 kinase, 70 kD, a member of the ribosomal protein S6 kinase(RSK) family of protein kinases, insulin and mitogen activated, andplays roles in cell cycle progression and control of cell proliferationGrove, J. R. et al., Cloning and expression of two human p70 S6 kinasepolypeptides differing only at their amino termini, Mol Cell Biol 11,5541-50 (1991). 11 7516613CD1 g1872546 0.0 [Mus musculus] NIK Su, Y. C.et al., NIK is a new Ste20-related kinase that binds NCK and MEKK1 andactivates the SAPK/JNK cascade via a conserved regulatory domain, EMBOJ. 16, 1279- 582239|Map4k4 0.0 [Mus musculus][Proteinkinase;Transferase;Receptor (signalling)] Mitogen-activated proteinkinase kinase kinase kinase 4, a serine-threonine kinase, interacts withNck, interacts with MEKK1 (Map3k1) and activates the c-Jun N-terminalkinase (Mapk8) signaling pathway; mutants fail to develop somites or ahindgut Becker, E. et al., Nck-interacting Ste20 kinase couples Ephreceptors to c-Jun N-terminal kinase and integrin activation, Mol CellBiol 20, 1537-45. (2000). 340694|MAP4K4 0.0 [Homo sapiens][Proteinkinase;Transferase] Mitogen-activated protein kinase kinase kinasekinase 4, a serine-threonine kinase, activates the c-Jun N-terminalkinase (MAPK8) signaling pathway, does not activate the ERK or p38(CSBP1) kinase pathways, may be involved in TNF-alpha (TNF) signalingYao, Z. et al., A novel human STE20-related protein kinase, HGK, thatspecifically activates the c-Jun N-terminal kinase signaling pathway, JBiol Chem 274, 2118-25 (1999). 12 7517068CD1 g6110362 0.0 [Homo sapiens]Traf2 and NCK interacting kinase, splice variant 7 Fu, C. A. et al.,TNIK, a novel member of the germinal center kinase family that activatesthe c-Jun N-terminal kinase pathway and regulates the cytoskeleton, I.Biol. Chem. 274, 30729-30737 (1999) 340694| 0.0 [Homo sapiens][Proteinkinase;Transferase] Mitogen-activated protein kinase kinase kinaseMAP4K4 kinase 4, a serine-threonine kinase, activates the c-JunN-terminal kinase (MAPK8) signaling pathway, does not activate the ERKor p38 (CSBP1) kinase pathways, may be involved in TNF-alpha (TNF)signaling Yao, Z. et al., A novel human STE20-related protein kinase,HGK, that specifically activates the c-Jun N-terminal kinase signalingpathway, J Biol Chem 274, 2118-25 (1999). 582239| 0.0 [Musmusculus][Protein kinase;Transferase;Receptor (signalling)]Mitogen-activated Map4k4 protein kinase kinase kinase kinase 4, aserine-threonine kinase, interacts with Nck, interacts with MEKK1(Map3k1) and activates the c-Jun N-terminal kinase (Mapk8) signalingpathway; mutants fail to develop somites or a hindgut Su, Y. C. et al.,NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activatesthe SAPK/JNK cascade via a conserved regulatory domain, Embo Journal 16,1279-90 (1997). 13 7517148CD1 g312395 0.0 [Homo sapiens] beta-adrenergickinase 2 Parruti, G. et al., Molecular cloning, functional expressionand mRNA analysis of human beta-adrenergic receptor kinase 2, Biochem.Biophys. Res. Commun. 190, 475-481 (1993) 341946| 0.0 [Homosapiens][Protein kinase;Transferase][Cytoplasmic;Plasma membrane]G-protein ADRBK2 coupled receptor kinase 3, member of a family ofprotein kinases that specifically phosphorylate activated G proteincoupled receptors, resulting in receptor desensitization, may representa genetic marker for mood disorders Parruti, G. et al., Molecularcloning, functional expression and mRNA analysis of humanbeta-adrenergic receptor kinase 2, Biochem Biophys Res Commun 190,475-81 (1993). 589791|Adrbk2 0.0 [Rattus norvegicus][Proteinkinase;Transferase][Axon;Dense bodies][G-protein coupled receptor kinase3, member of a family of protein kinases that specifically phosphorylateactivated G protein coupled receptors, resulting in receptordesensitization, may regulate nociception, sperm chemotaxis andolfaction Kovoor, A. et al., Agonist induced homologous desensitizationof mu-opioid receptors mediated by G protein-coupled receptor kinases isdependent on agonist efficacy, Mol Pharmacol 54, 704-11 (1998). 147517238CD1 g15559349 0.0 [Homo sapiens] Similar to likely ortholog ofmaternal embryonic leucine zipper kinase 570006|MELK 0.0 [Homosapiens][Protein kinase;Transferase] Protein containing two C-terminalkinase associated domain 1 and two protein kinase domains, has lowsimilarity to microtubule- MAP-affinity regulating kinase (ratLOC60328), which is a serine-threonine kinase that influencesmicrotubule stability Seong, H. A. et al., Phosphorylation of a novelzinc-finger-like protein, ZPR9, by murine protein serine/threoninekinase 38 (MPK38), Biochem J 361, 597-604. (2002). 585291|Melk  6.8E−270[Mus musculus]Protein kinase;Transferase] Protein containing a proteinkinase domain and a C-terminal kinase associated domain 1, has lowsimilarity to rat LOC60328, which is a serine-threonine kinase thatparticipates in microtubule stability and the control of cell polaritySeong, H. A. et al., Phosphorylation of a novel zinc-finger-likeprotein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38),Biochem J 361, 597-604. (2002). 15 7518685CD1 g4100632 0.0 [Homosapiens] lymphoid phosphatase LyP1 Cohen, S. et al., Cloning andcharacterization of a lymphoid-specific, inducible human proteintyrosine phosphatase, Lyp, Blood 93, 2013-2024 (1999) 570850| 0.0 [Homosapiens][Protein phosphatase;Hydrolase] Protein tyrosine phosphatasenon-receptor PTPN22 type 22, a protein tyrosine phosphatase that may beinvolved in T-cell development Cohen, S. et al., Cloning andcharacterization of a lymphoid-specific, inducible human proteintyrosine phosphatase, Lyp, Blood 93, 2013-24 (1999). 582663|  4.2E−270[Mus musculus][Protein phosphatase;Hydrolase] Protein tyrosinephosphatase non-receptor Ptpn8 type 8, a protein tyrosine phosphatasethat inhibits T-cell receptor mediated T-cell activation and is requiredfor B-cell antigen receptor-mediated growth arrest and apoptosisMatthews, R. J. et al., Characterization of hematopoietic intracellularprotein tyrosine phosphatases: description of a phosphatase containingan SH2 domain and another enriched in proline-, glutamic acid-, serine-,and threonine- rich sequences, Mol Cell Biol 12, 2396- 405 (1992). 167520192CD1 g190748  4.7E−105 [Homo sapiens] protein-tyrosine phophataseGu, M. X. et al., Identification, cloning, and expression of a cytosolicmegakaryocyte protein-tyrosine-phosphatase with sequence homology tocytoskeletal protein 4.1, Proc. Natl. Acad. Sci. U.S.A. 88, 5867-5871(1991) 337402|  3.3E−106 [Homo sapiens][Proteinphosphatase;Hydrolase][Cytoplasmic] Protein tyrosine phosphatase PTPN4non-receptor type 4, a non-membrane spanning protein tyrosinephosphatase that can inhibit cell proliferation, may play a role insignal transduction Gu, M. et al., The properties of the proteintyrosine phosphatase PTPMEG, J Biol Chem 271, 27751-9. (1996). 628499| 1.2E−102 [Mus musculus][Protein phosphatase;Hydrolase] Protein tyrosinephosphatase non-receptor Ptpn4 type 4, a protein tyrosine phosphatasethat may play a role in spermatogenesis Hironaka, K. et al., Theprotein-tyrosine phosphatase PTPMEG interacts with glutamate receptordelta 2 and epsilon subunits, J Biol Chem 275, 16167-73 (2000). 177520428CD1 g13537204 0.0 [Homo sapiens] MAST205 742582| 0.0 [Homosapiens][Protein kinase;Transferase][Cytoskeletal] Protein with strongsimilarity to MAST205 microtubule associated testis specificserine/threonine protein kinase (mouse Mtssk), which may act inspermatid maturation and microtubule organization, contains a proteinkinase domain and a PDZ, DHR, or GLGF domain Walden, P. D. et al.,Increased activity associated with the MAST205 protein kinase complexduring mammalian spermiogenesis, Biol Reprod 55, 1039-44 (1996).582149|Mtssk 0.0 [Mus musculus][Proteinkinase;Transferase][Cytoplasmic;Cytoskeletal] Microtubule associatedtestis specific serine/threonine protein kinase, may be involved in theorganization of manchette microtubules in spermatids, may have a role inspermatid maturation Walden, P. D. et al., Increased activity associatedwith the MAST205 protein kinase complex during mammalian spermiogenesis,Biol Reprod 55, 1039-44 (1996). 18 7522586CD1 g507162 6.7E−54 [Homosapiens] PITSLRE alpha 2-3 Xiang, J. et al., Molecular cloning andexpression of alternatively spliced PITSLRE protein kinase isoforms, J.Biol. Chem. 269, 15786-15794 (1994) 618480| 1.3E−53 [Homosapiens][Protein kinase;Transferase][Nuclear;Cytoplasmic] Cell divisioncycle 2 CDC2L1 like 1, member of the p34 (CDC2) superfamily thatcontains a PSTAIRE box, a protein kinase involved in apoptosis and cellcycle control; mutation of the corresponding gene is associated withnon-Hodgkin lymphoma and melanoma Lahti, J. M. et al., PITSLRE proteinkinase activity is associated with apoptosis, Mol Cell Biol 15, 1-11(1995). 581017| 2.9E−53 [Mus musculus][Protein kinase;Transferase] Celldivision cycle 2 like 2, a protein kinase Cdc2l2 that binds Src-homology2 (SH2) domains, appears to be involved in cell proliferation duringembryonic development, member of the p34(cdc2) superfamily Malek, S. N.et al., A cyclin-dependent kinase homologue, p130PITSLRE is aphosphotyrosine- independent SH2 ligand, J Biol Chem 269, 33009-20(1994). 19 7524017CD1 g1405935  4.4E−275 [Mus musculus] serine/threoninekinase Heyer, B. S. et al., New member of the Snf1/AMPK kinase family,Melk, is expressed in the mouse egg and preimplantation embryo, Mol.Reprod. Dev. 47, 148-156 (1997) 570006| 0.0 [Homo sapiens][Proteinkinase;Transferase] Protein containing two C-terminal kinase MELKassociated domain 1 and two protein kinase domains, has low similarityto microtubule- MAP-affinity regulating kinase (rat LOC60328), which isa serine-threonine kinase that influences microtubule stability Seong,H. A. et al., Phosphorylation of a novel zinc-finger-like protein, ZPR9,by murine protein serine/threonine kinase 38 (MPK38), Biochem J 361,597-604. (2002). 585291|Melk  3.0E−276 [Mus musculus][Proteinkinase;Transferase] Protein containing a protein kinase domain and aC-terminal kinase associated domain 1, has low similarity to ratLOC60328, which is a serine-threonine kinase that participates inmicrotubule stability and the control of cell polarity Gil, M. et al.,Cloning and expression of a cDNA encoding a novel proteinserine/threonine kinase predominantly expressed in hematopoietic cells,Gene 195, 295-301 (1997). 20 7525773CD1 g187561  1.1E−151 [Homo sapiens]mevalonate kinase Schafer, B. L. et al., Molecular cloning of humanmevalonate kinase and identification of a missense mutation in thegenetic disease mevalonic aciduria, J. Biol. Chem. 267, 13229- 13238(1992) 339520|MVK  7.9E−153 [Homo sapiens][Proteinkinase;Transferase;Other kinase] Mevalonate kinase (mevalonic aciduria),a peroxisomal enzyme involved in isoprenoid and cholesterolbiosynthesis; mutations in the corresponding gene cause mevalonicaciduria, hyperimmunoglobulinemia D and periodic fever syndrome Cho, Y.K. et al., Investigation of invariant serine/threonine residues inmevalonate kinase. Tests of the functional significance of a proposedsubstrate binding motif and a site implicated in human inheriteddisease, J Biol Chem 276, 12573-8. (2001). 704952|Mvk  1.8E−129 [Rattusnorvegicus][Transferase;Other kinase] Mevalonate kinase, a peroxisomalenzyme involved in isoprenoid and cholesterol biosynthesis; deficiencyof human MVK causes mevalonic aciduria, hyperimmunoglobulinemia D andperiodic fever syndrome Potter, D. et al., Identification and functionalcharacterization of an active-site lysine in mevalonate kinase, J BiolChem 272, 5741-6. (1997). 21 7525861CD1 g22328117 8.6E−77 [Homo sapiens]similar to protein-tyrosine-phosphatase homolog DKFZp566K0524.1— human(fragment) 425672| 1.1E−83 [Homo sapiens][Proteinphosphatase;Hydrolase][Cytoplasmic] Protein with high similarityDKFZP566- to protein tyrosine phosphatase non-receptor type 20 (mousePtpn20), which is a testis- K0524 specific protein tyrosine phosphatasethat may play a role in spermatogenesis or meiosis, member of theprotein-tyrosine phosphatase family 582661| 2.5E−47 [Musmusculus][Protein phosphatase;Hydrolase][Cytoplasmic] Protein tyrosinephosphatase Ptpn20 non-receptor type 20, a testis-specific proteintyrosine phosphatase that may play a role in spermatogenesis or meiosisOhsugi, M. et al., Molecular cloning and characterization of a novelcytoplasmic protein- tyrosine phosphatase that is specifically expressedin spermatocytes, J Biol Chem 272, 33092-9 (1997). 22 2509577CD1g10312094 6.4E−40 NIMA-related serine/threonine kinase [Mus musculus]Kandli, M. et al. Isolation and characterization of two evolutionarilyconserved murine kinases (Nek6 and nek7) related to the fungal mitoticregulator, NIMA. Genomics 68, 187- 196 (2000) 2509577CD1 714317| 2.2E−130 [Caenorhabditis elegans] Protein containing 16 EB moduledomains and a protein kinase D1044.3 domain, has a region of lowsimilarity to NIMA (never in mitosis gene a) -related expressed kinase 6(human NEK6), which activates the S6 ribosomal protein kinase p70S6K(RPS6KB1) Chervitz, S. A. et al. Comparison of the complete protein setsof worm and yeast: Orthology and divergence. Science 282, 2022-2028(1998) 2509577CD1 789751|NEK6 1.3E−40 [Homo sapiens][Proteinkinase;Transferase] NIMA (never in mitosis gene a) -related expressedkinase 6, a protein kinase that phosphorylates and activates the S6ribosomal protein kinase p70S6K (RPS6KB1) Belham, C. et al.Identification of the NIMA family kinases NEK6/7 as regulators of thep70 ribosomal S6 kinase. Curr. Biol. 11, 1155-1167 (2001) 23 7505222CD1g256855  5.4E-112 serine/threonine- and tyrosine-specific proteinkinase; Nek1 [Mus sp.] Letwin, K. et al. A mammalian dual specificityprotein kinase, Nek1, is related to the NIMA cell cycle regulator andhighly expressed in meiotic germ cells. EMBO J. 11, 3521- 3531 (1992)7505222CD1 750718|NEK1 −2.3E−110 [Homo sapiens] Protein containing aprotein kinase domain, has weak similarity to serine threonine kinase 2(mouse Stk2), which undergoes cleavage by caspase 3 (mouse Casp3) andthe released N-terminal kinase domain and C-terminal domain promoteapoptosis Nagase, T. et al. Prediction of the coding sequences ofunidentified human genes. XXI. The complete sequences of 60 new cDNAclones from brain which code for large proteins. DNA Res. 8, 179-187(2001) 7505222CD1 430066|Nek3 2.0E−92 [Mus musculus][Proteinkinase;Transferase][Cytoplasmic] NIMA-related kinase 3, a protein kinasethat is involved in cell cycle control Chen, A. et al. NIMA-relatedkinases: isolation and characterization of murine nek3 and nek4 cDNAs,and chromosomal localization of nek1, nek2 and nek3. Gene 234, 127-137(1999) 24 7524408CD1 g4583675 3.7E−264 [Homo sapiens] apyraseBiederbick, A. et al. A human intracellular apyrase-like protein,LALP70, localizes to lysosomal/autophagic vacuoles. J. Cell. Sci. 112(Pt 15), 2473-2484 (1999) 7524408CD1 340820|LYSAL1  2.7E−265 [Homosapiens][Other phosphatase;Hydrolase][Lysosome/vacuole;Cytoplasmic]Lysosomal apyrase-like protein (Golgi apyrase), a member of the apyraseor GDA1/CD39 family that is a lysosomal membrane protein with fourapyrase domains, alternative splice form is identical to uridinediphosphatase Biederbick, A. et al. First apyrase splice variants havedifferent enzymatic properties. Biol. Chem. 275, 19018-19024 (2000)7524408CD1 753913|Lysal2  2.8E−160 [Mus musculus] Protein with highsimilarity to lysosomal apyrase-like protein (Golgi apyrase, humanLYSAL1), which is a lysosomal membrane protein with four apyrasedomains, member of the GDA1 or CD39 family of nucleoside phosphatasesShi, J. D. et al. Molecular cloning and characterization of a novelmammalian endo-apyrase (LALP1). J. Biol. Chem. 276, 17474-17478 (2001)7526163CD1 423529| 0.0 [Homo sapiens][Protein kinase;Transferase]Protein with high similarity to murine Mtssk, KIAA0561 which is aprotein kinase that interacts with microtubules and facilitates theirorganization in spermatids, contains a eukaryotic protein kinase domainand a PDZ domain Nagase, T. Prediction of the coding sequences ofunidentified human genes. IX. The complete sequences of 100 new cDNAclones from brain which can code for large proteins in vitro. DNA Res.5, 31-39 (1998) 7526163CD1 742582| 0.0 [Homo sapiens][Proteinkinase;Transferase][Cytoskeletal] Protein with strong similarity toMAST205 microtubule associated testis specific serine/threonine proteinkinase (mouse Mtssk), which may act in spermatid maturation andmicrotubule organization, contains a protein kinase domain and a PDZ,DHR, or GLGF domain Walden, P. D. et al. Increased activity associatedwith the MAST205 protein kinase complex during mammalian spermiogenesis.Biol. Reprod. 55, 1039-1044 (1996) 26 7526158CD1 g406058 0.0 proteinkinase [Mus musculus] Walden, P. D. et al. A novel 205-kilodaltontestis-specific serine/threonine protein kinase associated withmicrotubules of the spermatid manchette. Mol. Cell. Biol. 13, 7625-7635(1993) 7526158CD1 423529| 0.0 [Homo sapiens][Protein kinase;Transferase]Protein with high similarity to murine Mtssk, KIAA0561 which is aprotein kinase that interacts with microtubules and facilitates theirorganization in spermatids, contains a eukaryotic protein kinase domainand a PDZ domain Nagase, T. DNA Res. 5, 31-39 (1998) supra 7526158CD1582149|Mtssk 0.0E+00 [Mus musculus][Proteinkinase;Transferase][Cytoplasmic;Cytoskeletal] Microtubule associatedtestis specific serine/threonine protein kinase, may be involved in theorganization of manchette microtubules in spermatids, may have a role inspermatid maturation Lumeng, C. et al. Interactions between beta2-syntrophin and a family of microtubule- associated serine/threoninekinases. Nat. Neurosci. 2, 611-617 (1999) 27 7519807CD1 g181489115.7E−35 [Homo sapiens]SKRP1 Zama, T. et al., A novel dual specificityphosphatase SKRP1 interacts with the MAPK kinase MKK7 and inactivatesthe JNK MAPK pathway. Implication for the precise regulation of theparticular MAPK pathway, J. Biol. Chem. 277, 23909-23918 (2002) Zama, T.et al., Scaffold role of a mitogen-activated protein kinase phosphatase,SKRP1, for the JNK signaling pathway, J. Biol. Chem. 277, 23919-23926(2002) 773093| 3.4E−36 [Homo sapiens] Protein containing two dualspecificity phosphatase catalytic domains, has SKRP1 moderate similarityto dual specificity phosphatase 3 (vaccinia H1 related phosphatase,human DUSP3), which dephosphorylates phosphotyrosine and phosphoserine,and inactivates MAPK 28 7526180CD1 g8250239  1.4E−241 proteinphosphatase 4 regulatory subunit 2 [Homo sapiens] Hastie, C. J. et al.,A novel 50 kDa protein forms complexes with protein phosphatase 4 and islocated at centrosomal microtubule organizing centres, Biochem. J. 347Pt 3, 845-855 (2000) 7526180CD1 606258|PPP4R2  7.8E−243 [Homosapiens][Regulatory subunit][Cytoplasmic;Centrosome/spindle pole body]Protein phosphatase 4 regulatory subunit 2, interacts with proteinphosphatase 4 catalytic subunit (PPP4C), may target PPP4C to thecentrosome and regulate its activity at centrosomal microtubuleorganizing centers Hastie, C. J. et al. (supra) 29 7526185CD1 g25824138.0E−74 STE20-like kinase 3 [Homo sapiens] Schinkmann, K. A. et al.,Cloning and characterization of a novel mammalian STE20-like kinase(mst-3), J. Biol. Chem. 272, 286995-286703 (1997) 7526185CD1336486|STK24 4.4E−75 [Homo sapiens][Protein kinase;Transferase]Serine-threonine kinase 24 (Ste20 yeast homolog), member of the SPS1subgroup of the STE20-like protein family, a serine- threonine kinasethat prefers manganese as a cofactor and uses either GTP or ATP as aphosphate donor Zhou, T. H. et al., Identification of a humanbrain-specific isoform of mammalian STE20- like kinase 3 that isregulated by cAMP-dependent protein kinase., J Biol Chem 275, 2513- 9(2000). 7526185CD1 743574|MST4 4.0E−65 [Homo sapiens][Proteinkinase;Transferase] Mst3 and SOK1-related kinase, a protein kinase,induces apoptosis, involved in cell growth, appears to activate MAPK butnot JNK nor p38 kinase pathways, alternative form MST4a may regulateMST4; gene maps to a region associated with mental retardation Lin, J.L. et al., MST4, a new Ste20-related kinase that mediates cell growthand transformation via modulating ERK pathway. Oncogene 20, 6559-69.(2001). 30 7526192CD1 g2199529  1.5E−134 casein kinase I gamma 2 [Homosapiens] Kitabayashi, A. N. et al., Cloning and chromosomal mapping ofhuman casein kinase I gamma 2 (CSNK1G2), Genomics 46, 133-137 (1997)7526192CD1 344104|  8.1E−136 [Homo sapiens][Protein kinase;Transferase]Casein kinase 1 gamma 2, a putative CSNK1G2 serine/threonine proteinkinase, may play a role in signal transduction Kitabayashi, A. N. etal., Cloning and chromosomal mapping of human casein kinase I gamma 2(CSNK1G2)., Genomics 46, 133-7 (1997). 7526192CD1 664931|  2.9E−129[Rattus norvegicus][Protein kinase;Transferase] Casein kinase 1 gamma 2,serine/threonine Csnk1g2 protein kinase, may play a role in receptortyrosine kinase-mediated signal transduction Voisin, L. et al.,Angiotensin II stimulates serine phosphorylation of the adaptor proteinNck: physical association with the serine/threonine kinases Paid andcasein kinase I., Biochem J 341, 217-23 (1999). 31 7526193CD1 g15215576 1.1E−166 BMP-2 inducible kinase [Mus musculus] Kearns, A. E. et al.,Cloning and characterization of a novel protein kinase that impairsosteoblast differentiation in vitro, J. Biol. Chem. 276, 42213-42218(2001) 7526193CD1 770160|Bike  6.1E-168 [Mus musculus] Proteincontaining a protein kinase domain, has low similarity to C. elegansSEL-5, which is a serine-threonine protein kinase that likely regulatesLIN-12 and GLP-1 signaling Kearns, A. E. et al. (supra) 7526193CD1244458|sel-5 1.2E−60 [Caenorhabditis elegans][Proteinkinase][Cytoplasmic] Serine/threonine protein kinase which likelyregulates LIN-12 and GLP-1 signaling; has similarity to S. cerevisiaeArk1p and Prk1p protein kinases which are involved in regulation of thecytoskeleton Fares, H. et al., SEL-5, A Serine/Threonine Kinase ThatFacilitates lin-12 Activity in Caenorhabditis elegans., Genetics 153,1641-1654 (1999). 32 7526196CD1 g2506080 4.5E−40 HsGAK [Homo sapiens]Kimura, S. H. et al., Structure, expression, and chromosomallocalization of human GAK, Genomics 44, 179-187 (1997) 7526196CD1342050|GAK 2.5E−41 [Homo sapiens][Protein kinase;Transferase] CyclinG-associated kinase, a putative serine/threonine protein kinase thatshares homology with tensin and auxilin, may play a role in cell cycleregulation Kimura, S. H. et al. (supra) 7526196CD1 704892|Gak 1.1E−40[Rattus norvegicus][Protein kinase;Transferase] Cyclin G-associatedkinase, a serine/threonine protein kinase that shares homology withtensin and auxilin, interacts with cyclin G (Ccng1)- Cdk5 complex,involved in the dissociation of clathrin-coated vesicles in non-neuronalcells Greener, T. et al., Role of cyclin G-associated kinase inuncoating clathrin-coated vesicles from non-neuronal cells., J Biol Chem275, 1365-70. (2000). 33 7526198CD1 g2506080 0.0 HsGAK [Homo sapiens]Kimura, S. H. et al. (supra) 7526198CD1 342050|GAK 0.0 [Homosapiens][Protein kinase;Transferase] Cyclin G-associated kinase, aputative serine/threonine protein kinase that shares homology withtensin and auxilin, may play a role in cell cycle regulation Kimura, S.H. et al. (supra) 7526198CD1 704892|Gak 0.0 [Rattus norvegicus][Proteinkinase;Transferase] Cyclin G-associated kinase, a serine/threonineprotein kinase that shares homology with tensin and auxilin, interactswith cyclin G (Ccng1)- Cdk5 complex, involved in the dissociation ofclathrin-coated vesicles in non-neuronal cells Greener, T. et al.(supra) 34 7526208CD1 g4426595  9.0E−255 multifunctionalcalcium/calmodulin-dependent protein kinase II delta2 isoform [Homosapiens] Hoch, B. et al., Identification and expression ofdelta-isoforms of the multifunctional Ca2+/calmodulin-dependent proteinkinase in failing and nonfailing human myocardium, Circ. Res. 84,713-721 (1999) 7526208CD1 742886|  4.9E−256 [Homo sapiens][Proteinkinase;Transferase][Nuclear;Cytoplasmic] Calcium/calmodulin- CAMK2Ddependent protein kinase II delta, member of the multifunctional CAMKIIfamily involved in Ca2+ regulated processes; alternative form delta 3 isspecifically upregulated in the myocardium of patients with heartfailure Hoch, B. et al.(supra) 7526208CD1 772372|  3.1E−243 [Musmusculus] Protein with strong similarity to calcium-calmodulin-dependentprotein Camk2d kinase II delta (rat Camk2d), which is involved in Ca2+regulated processes, contains two protein kinase domains Hoch, B. etal., delta-Ca(2+)/calmodulin-dependent protein kinase II expressionpattern in adult mouse heart and cardiogenic differentiation ofembryonic stem cells, J Cell Biochem 79, 293-300 (2000). 35 7526212CD1g1661132  5.3E−169 calcium/calmodulin-dependent protein kinase II delta2-subunit [Sus scrofa] Singer, H. A. et al., NovelCa2+/calmodulin-dependent protein kinase II gamma-subunit variantsexpressed in vascular smooth muscle, brain, and cardiomyocytes, J. Biol.Chem. 272, 9393-9400 (1997) 7526212CD1 772372|Camk2d  2.9E−170 [Musmusculus] Protein with strong similarity to calcium-calmodulin-dependentprotein kinase II delta (rat Camk2d), which is involved in Ca2+regulated processes, contains two protein kinase domains Hoch, B. etal., J Cell Biochem 79, 293-300 (2000). (supra) 7526212CD1 742886| 1.6E−169 [Homo sapiens][Proteinkinase;Transferase][Nuclear;Cytoplasmic] Calcium/calmodulin- CAMK2Ddependent protein kinase II delta, member of the multifunctional CAMKIIfamily involved in Ca2+ regulated processes; alternative form delta 3 isspecifically upregulated in the myocardium of patients with heartfailure Hoch, B. et al., Circ Res 84, 713-21. (1999). (supra) 367526213CD1 g15215576 2.1E−15 BMP-2 inducible kinase [Mus musculus]Kearns, A. E. et al. (supra) 7526213CD1 605792|BIKE 1.7E−27 [Homosapiens][Protein kinase;Transferase] Protein containing a eukaryoticprotein kinase domain 7526213CD1 770160|Bike 1.1E−16 [Mus musculus]Protein containing a protein kinase domain, has low similarity to C.elegans SEL-5, which is a serine-threonine protein kinase that likelyregulates LIN-12 and GLP-1 signaling Kearns, A. E. et al. (supra) 377526214CD1 g15215576 1.7E−16 BMP-2 inducible kinase [Mus musculus]Kearns, A. E. et al. (supra) 7526214CD1 605792|BIKE 3.8E−28 [Homosapiens][Protein kinase;Transferase] Protein containing a eukaryoticprotein kinase domain 7526214CD1 770160|Bike 9.4E−18 [Mus musculus]Protein containing a protein kinase domain, has low similarity to C.elegans SEL-5, which is a serine-threonine protein kinase that likelyregulates LIN-12 and GLP-1 signaling Kearns, A. E. et al. (supra) 387526228CD1 g2924624 4.6E−55 TGF-beta activated kinase 1a [Homo sapiens]Sakurai, H. et al., TGF-beta-activated kinase 1 stimulates NF-kappa Bactivation by an NF- kappa B-inducing kinase-independent mechanism,Biochem. Biophys. Res. Commun. 243, 545-549 (1998) 7526228CD1 338400|2.5E−56 [Homo sapiens][Protein kinase;Transferase] Mitogen-activatedprotein kinase kinase kinase MAP3K7 7 (TGF beta activated kinase 1),mediates TGFbeta and IL1 signal transduction, induces NFkappaBactivation, may act as a regulatory kinase of I kappa B kinases (IKKs)Sakurai, H. et al., Functional interactions of transforming growthfactor beta-activated kinase 1 with IkappaB kinases to stimulateNF-kappaB activation., J Biol Chem 274, 10641-8 (1999). 7526228CD1338400| 2.50E−56 [Homo sapiens][Protein kinase;Transferase]Mitogen-activated protein kinase kinase MAP3K7 kinase 7 (TGF betaactivated kinase 1), mediates TGFbeta and IL1 signal transduction,induces NFkappaB activation, may act as a regulatory kinase of I kappa Bkinases (IKKs) Craig, R. et al., p38 MAPK and NF-kappa B collaborate toinduce interleukin-6 gene expression and release. Evidence for acytoprotective autocrine signaling pathway in a cardiac myocyte modelsystem., J Biol Chem 275, 23814-24 (2000). 39 7526246CD1 g232727395.7E−96 adrenergic, beta, receptor kinase 1 [Homo sapiens] Strausberg,R. L. et al., Generation and initial analysis of more than 15,000full-length human and mouse cDNA sequences, Proc. Natl. Acad. Sci.U.S.A. 99, 16899-16903 (2002) 7526246CD1 334086| 3.1E−97 [Homosapiens][Protein kinase;Transferase][Cytoplasmic;Plasma membrane] Beta-ADRBK1 adrenergic receptor kinase 1, kinase that mediatesdesensitization of G protein-coupled receptors, phosphorylated by PKC,may modulate cardiovascular function; mouse and rat Adrbk1 appear to beinvolved with cardiomyopathy and myocardial infarction Shih, M. et al.,Oligodeoxynucleotides antisense to mRNA encoding protein kinase A,protein kinase C, and beta-adrenergic receptor kinase reveal distinctivecell-type-specific roles in agonist-induced desensitization., Proc NatlAcad Sci USA 91, 12193-7 (1994). 7526246CD1 775647|Adrbk1 1.1E−94 [Musmusculus][Protein kinase;Transferase] Beta-adrenergic receptor kinase 1,a kinase that may mediate desensitization of G protein-coupledreceptors, modulates myocardial function and involved in cardiomyopathy;human ADRBK1 may play roles in hypertension and cardiomyopathy Proll, M.A. et al., Beta 2-adrenergic receptor mutants reveal structuralrequirements for the desensitization observed with long-term epinephrinetreatment., Mol Pharmacol 44, 569-74 (1993). 40 7526258CD1 g33303889 9.6E−110 FAST kinase [synthetic construct] 7526258CD1 743544|FASTK 5.2E−111 [Homo sapiens][Protein kinase;Transferase] Fas-activatedserine threonine kinase, a serine- threonine kinase that phosphorylatesRNA binding protein TIAl during Fas mediated apoptosis, upregulated inperipheral blood mononuclear cells of atopic asthmatics and atopic nonasthmatic patients Brutsche, M. H. et al., Apoptosis signals in atopyand asthma measured with cDNA arrays., Clin Exp Immunol 123, 181-7.(2001). 7526258CD1 685389| 1.6E−11 [Homo sapiens] Protein of unknownfunction, has a region of low similarity to a region of MGC5297fas-activated serine threonine kinase (human FASTK), which is aserine-threonine kinase that phosphorylates RNA binding protein humanTIA1 during Fas mediated apoptosis 41 7526311CD1 g1088281 7.9E−67pyruvate dehydrogenase kinase [Homo sapiens] Gudi, R. et al., Diversityof the pyruvate dehydrogenase kinase gene family in humans, J. Biol.Chem. 270, 28989-28994 (1995) 7526311CD1 336846|PDK1 4.3E−68 [Homosapiens][Protein kinase;Transferase;Otherkinase][Cytoplasmic;Mitochondrial] Pyruvate dehydrogenase kinase 1,phosphorylates and inactivates the pyruvate dehydrogenase complex andthus regulates pyruvate metabolism Taylor, V. et al., 5′ phospholipidphosphatase SHIP-2 causes protein kinase B inactivation and cell cyclearrest in glioblastoma cells., Mol Cell Biol 20, 6860-71 (2000).7526311CD1 757382|Pdk1 2.2E−55 [Rattus norvegicus][Proteinkinase;Transferase;Other kinase][Cytoplasmic;Mitochondrial] Pyruvatedehydrogenase kinase 1, phosphorylates and inactivates the pyruvatedehydrogenase complex and thus putatively regulates pyruvate metabolismSugden, M. C. et al., Expression and regulation of pyruvatedehydrogenase kinase isoforms in the developing rat heart and inadulthood: role of thyroid hormone status and lipid supply, Biochem J352, 731-8. 2000). 42 7526315CD1 g12655099  7.2E−121 Mixed lineagekinase-related kinase MRK-beta [Homo sapiens] Strausberg, R. L. et al.(supra) 7526315CD1 476453|ZAK  3.9E−122 [Homo sapiens] Mixed lineagekinase with a leucine zipper and a sterile alpha motif, a mixed lineagekinase-like protein that stimulates the JNK/SAPK pathway and activatesNF- kappaB; overexpression induces apoptosis of a hepatoma cell lineLiu, T. C. et al., Cloning and expression of ZAK, a mixed lineagekinase-like protein containing a leucine-zipper and a sterile-alphamotif, Biochem Biophys Res Commun 274, 811-6 (2000). 7526315CD1662697|Zak  2.7E−121 [Mus musculus][Protein kinase;Transferase] Mixedlineage kinase with a leucine zipper and a sterile alpha motif,activated by osmotic shock; overexpression activates the p38 (Mapk14),JNK/SAPK, ERK (Mapk3), and ERK5 (Mapk7) pathways, alpha alternative formdisrupts actin stress fibers Gotoh, I. et al., Identification andcharacterization of a novel MAP kinase kinase kinase, MLTK., J Biol Chem276, 4276-86 (2001). (supra) 43 7526442CD1 g12803641 3.5E−64 CCRKprotein [Homo sapiens] Strausberg, R. L. et al. (supra) 7526442CD1568698|CCRK 2.4E−65 [Homo sapiens][Protein kinase;Transferase] Proteincontaining four protein kinase domains, has a region of moderatesimilarity to cyclin-dependent kinase 3 (human CDK3), which is a kinasethat binds to cyclin A and is required for progression from G1 to Sphase 7526442CD1 583769|Cdk5 1.6E|22 [Mus musculus][Proteinkinase;Transferase][Cell body (soma);Growth cone] Cyclin- dependentprotein kinase 5, serine-threonine kinase that associates with theregulatory subunit p35 (Cdk5r) and phosphorylates neuronal proteins,involved in neuronal differentiation, regulation of myogenesis, andadaptive responses to cocaine Ohshima, T. et al., Targeted disruption ofthe cyclin-dependent kinase 5 gene results in abnormal corticogenesis,neuronal pathology and perinatal death., Proc Natl Acad Sci US A 93,11173-8 (1996).

TABLE 3 SEQ Incyte Amino ID Polypeptide Acid Analytical Methods NO: IDResidues Signature Sequences, Domains and Motifs and Databases 17517831CD1 83 signal_cleavage: M1-T58 SPSCAN KINASE TYROSINE-PROTEINPROTO-ONCOGENE DOMAIN TRANSFERASE ATP- BLAST_PRODOM BINDING MYRISTATEPHOSPHORYLATION SH2 SH3 PD012180: G2-E43 Potential PhosphorylationSites: S7 MOTIFS Potential Glycosylation Sites: N40, N67 MOTIFS 27520272CD1 292 signal_cleavage: M1-A44 SPSCANFructose-1-6-bisphosphatase: N12-H289 HMMER_PFAM Inositolphosphatase/fructose-1,6-bisphosphatase 1PB000146: G59-D100, G112-T135,Q155- BLIMPS_BLOCKS P189, R198-P220, G228-G253Fructose-1-6-bisphosphatase active site: H208-E255 PROFILESCANFructose-1,6-bisphosphatase signature PR00115: D119-Y140, P156-L176,G181-G196, A197- BLIMPS_PRINTS P220, G228-G248, V257-V279 Inositolphosphatase/fructose-1,6-bisphosphatase family signature PR00377:V115-N126, L211- BLIMPS_PRINTS A221, Y234-G248 HYDROLASE CARBOHYDRATEMETABOLISM FRUCTOSE-16-BISPHOSPHATASE BLAST_PRODOM FBPASE1-PHOSPHOHYDROLASE D-FRUCTOSE-1,6-BISPHOSPHATE CYCLE CHLOROPLAST CALVINPD001491:G68-P189 D188-V279 FRUCTOSE1 6-BISPHOSPHATASE1-PHOSPHOHYDROLASE FBPASE HYDROLASE BLAST_PRODOM CARBOHYDRATE METABOLISMD-FRUCTOSE-1, 6-BISPHOSPHATE MULTI-GENE PD017713: T13-V66FRUCTOSE-1-6-BISPHOSPHATASE BLAST_DOMO DM00535|P09467|10-331:V11-P189P189-E287 FRUCTOSE-1-6-BISPHOSPHATASE BLAST_DOMODM00535|A37295|60-331:A61-P189 P189-E287 FRUCTOSE-1-6-BISPHOSPHATASEBLAST_DOMO DM00535|S46245|11-332:T13-P189 D190-G274FRUCTOSE-1-6-BISPHOSPHATASE BLAST_DOMO DM00535|P46267|12-333:T13-P189D190-Y286 Potential Phosphorylation Sites: S97, S125, S144, S149, S275,T145, T252 MOTIFS Potential Glycosylation Sites: N65 MOTIFSFructose-1-6-bisphosphatase active site: G228-A240 MOTIFS 3 7521279CD1434 signal_cleavage: M1-G51 SPSCAN 6-phosphofructo-2-kinase: Q30-P249HMMER_PFAM Phosphoglycerate mutase family: R250-I400 HMMER_PFAMPhosphoglycerate mutase family IPB001345: 1252-S284, V299-A311,G315-R347 BLIMPS_BLOCKS Phosphoglycerate mutase family phosphohistidinesignature: I234-K283 PROFILESCAN 6-phosphofructo-2-kinase familysignature PR00991: V125-A139, K151-I165, P177-F191, BLIMPS_PRINTSV230-S251, I252-L274 MUTASE PROTEOME COMPLETE PHOSPHOGLYCERATE PGAMISOMERASE BLAST_PRODOM GLYCOLYSIS BPG-DEPENDENTFRUCTOSE-2,6-BISPHOSPHATASE PHOSPHOGLYCEROMUTASE PD000730:Y253-D328S330-L388 KINASE FRUCTOSE-2,6-BISPHOSPHATASE INCLUDES: ISOZYME6PF-2-K/FRU- 6- BLAST_PRODOMPHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BIPHOSPHATASE TRANSFERASE 2,6- P2ASEMULTI-FUNCTIONAL PD002665: T36-1252 6-BISPHOSPHATASE TRANSFERASE6PF2K/FRU2 6-P2ASE INCLUDES: KINASE BLAST_PRODOM FRUCTOSE2MULTI-FUNCTIONAL ENZYME ISOZYME PD009472: T389-H433 6-PF2K/FRU2,6-P2ASETESTIS ISOZYME INCLUDES: 6-PHOSPHOFRUCTO 2-KINASE BLAST_PRODOM EC2.7.1.105 FRUCTOSE-2,6-BISPHOSPHATASE 3.1.3.46 MULTI-FUNCTIONAL ENZYMETRANSFERASE KINASE HYDROLASE ATP-BINDING PD114268: M1-M356-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATE 2-PHOSPHATASEBLAST_DOMO DM01656|JC1470|184-441:E186-R337 Y331-V4076-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATE 2-PHOSPHATASEBLAST_DOMO DM01656|JC2037|185-444:N181-L352 S330-V4076-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATE 2-PHOSPHATASEBLAST_DOMO DM01656|P07953|184-442: G46-V82 D183-A329 S330-V4076-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATE 2-PHOSPHATASEBLAST_DOMO DM01656|P25114|183-441:D183-E349 S330-V407 PotentialPhosphorylation Sites: S3, S56, S204, S275, S330, T60, T85, T133, T140,T248, MOTIFS T409, Y377 Potential Glycosylation Sites: N132 MOTIFSATP/GTP-binding site motif A (P-loop): G46-T53 MOTIFS Phosphoglyceratemutase family phosphohistidine signature: L254-N263 MOTIFS 4 7523965CD1240 Phosphoenolpyruvate carboxykinase (GTP) IPB000364: K88-P121,F148-L178, T179-L202, BLIMPS_BLOCKS D204-P217 PHOSPHOENOLPYRUVATECARBOXYKINASE LYASE GTP-BINDING CARBOXYLASE BLAST_PRODOM DECARBOXYLASEGLUCONEOGENESIS PD004738: D46-E232 PHOSPHOENOLPYRUVATE CARBOXYKINASE,MITOCHONDRIAL PRECURSOR GTP BLAST_PRODOM EC 4.1.1.32 CARBOXYLASE PEPCKMGLUCONEOGENESIS LYASE DECARBOXYLASE GTP-BINDING MITOCHONDRION TRANSITPEPTIDE MANGANES PD144568: M1-R45 PHOSPHOENOLPYRUVATE CARBOXYKINASE(GTP) BLAST_DOMO DM01781|P05153|15-621: V32-P240 DM01781|P20007|40-646:G35-E232 DM01781|P21642|33-639: L33-P240 DM01781|Q05893|30-640: V32-E232Potential Phosphorylation Sites: S23, S51, S115, S136, S187, T29, T66,T75, T219 MOTIFS 5 7524016CD1 199 signal_cleavage: M1-T33 SPSCAN6-phosphofructo-2-kinase: R7-W199 HMMER_PFAM 6-phosphofructo-2-kinasefamily signature PR00991: V104-S118, K130-I144, P156-F170 BLIMPS_PRINTSKINASE FRUCTOSE-2,6-BISPHOSPHATASE INCLUDES: ISOZYME 6PF-2-K/FRU- 6-BLAST_PRODOM PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BIPHOSPHATASETRANSFERASE 2,6- P2ASE MULTI-FUNCTIONAL PD002665: W45-W194, A10-A1246-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATE 2-PHOSPHATASEBLAST_DOMO DM01457|JC1470|28-182: A10-C161 DM01457|P07953|29-182:A10-C161 DM01457|P25114|27-181: T16-D160 DM01457|P26285|26-180: T16-Y158Potential Phosphorylation Sites: S36, S64, S98, T5, T112 MOTIFSPotential Glycosylation Sites: N111 MOTIFS ATP/GTP-binding site motif A(P-loop): G26-T33 MOTIFS 6 7524680CD1 406 6-phosphofructo-2-kinase:M1-P186 HMMER_PFAM Phosphoglycerate mutase family: R187-I372 HMMER_PFAMPhosphoglycerate mutase family IPB001345: I189-A221, V236-A248,G252-E284, E301-E346 BLIMPS_BLOCKS Phosphoglycerate mutase familyphosphohistidine signature: I171-Y220 PROFILESCAN6-phosphofructo-2-kinase family signature PR00991: V62-S76, K88-I102,P114-F128, V167- BLIMPS_PRINTS S188, I189-L211, A266-P282 MUTASEPROTEOME COMPLETE PHOSPHOGLYCERATE PGAM ISOMERASE BLAST_PRODOMGLYCOLYSIS BPG-DEPENDENT FRUCTOSE-2,6-BISPHOSPHATASEPHOSPHOGLYCEROMUTASE PD000730:Y190-Y303 P298-L360 KINASEFRUCTOSE-2,6-BISPHOSPHATASE INCLUDES: ISOZYME 6PF-2-K/FRU- 6-BLAST_PRODOM PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BTPHOSPHATASETRANSFERASE 2,6- P2ASE MULTI-FUNCTIONAL PD002665: K9-I1896-BISPHOSPHATASE TRANSFERASE 6PF2K/FRU2 6-P2ASE INCLUDES: KINASEBLAST_PRODOM FRUCTOSE2 MULTI-FUNCTIONAL ENZYME ISOZYME PD009472:T361-Y406 FRUCTOSE-2 SIMILAR PD114271: S232-V376 BLAST_PRODOM6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATE 2-PHOSPHATASEBLAST_DOMO DM01656|JC1470|184-441: K123-V379 DM01656|P07953|184-442:D120-V379 DM01656|P25114|183-441: D120-V379 DM01656|P26285|182-441:D120-V379 Potential Phosphorylation Sites: S22, S56, S212, S233, S302,S343, T5, T70, T185, T273, T381, MOTIFS Y295, Y349 PotentialGlycosylation Sites: N69 MOTIFS Phosphoglycerate mutase familyphosphohistidine signature: L191-N200 MOTIFS 7 7524757CD1 426signal_cleavage: M1-T33 SPSCAN 6-phosphofructo-2-kinase: R7-P206HMMER_PFAM Phosphoglycerate mutase family: R207-I392 HMMER_PFAMPhosphoglycerate mutase family phosphohistidine signature: I191-Y240PROFILESCAN Phosphoglycerate mutase family IPB001345A: I209-A241,V256-A268, G272-E304, E321-E366 BLIMPS_BLOCKS 6-phosphofructo-2-kinasefamily signature PR00991: V82-S96, K108-I122, P134-F148, BLIMPS_PRINTSV187-S208, I209-L231, A286-P302 KINASE FRUCTOSE-2,6-BISPHOSPHATASEINCLUDES: ISOZYME 6PF-2-K/FRU- 6- BLAST_PRODOMPHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BIPHOSPHATASE TRANSFERASE 2,6- P2ASEMULTI-FUNCTIONAL PD002665:A10-A64 D52-I209 MUTASE PROTEOME COMPLETEPHOSPHOGLYCERATE PGAM ISOMERASE BLAST_PRODOM GLYCOLYSIS BPG-DEPENDENTFRUCTOSE-2,6-BISPHOSPHATASE PHOSPHOGLYCEROMUTASE PD000730:Y210-Y323P318-L380 6BISPHOSPHATASE TRANSFERASE 6PF2K/FRU2 6-P2ASE INCLUDES:KINASE BLAST_PRODOM FRUCTOSE2 MULTI-FUNCTIONAL ENZYME ISOZYME PD009472:T381-Y426 FRUCTOSE-2 SIMILAR PD114271: S252-V396 BLAST_PRODOM6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATE 2-PHOSPHATASEBLAST_DOMO DM01656|JC1470|184-441: K143-V399 DM01656|JC2037|185-444:D140-V399 DM01656|P07953|184-442: D140-V399 DM01656|P25114|183-441:D140-V399 Potential Phosphorylation Sites: S36, S76, S232, S253, S322,S363, T5, T90, T205, T293, T401, MOTIFS Y315, Y369 PotentialGlycosylation Sites: N89 MOTIFS ATP/GTP-binding site motif A (P-loop):G26-T33 MOTIFS Phosphoglycerate mutase family phosphohistidinesignature: L211-N220 MOTIFS 8 7516229CD1 355 signal_cleavage: M1-S48SPSCAN Phosphatidylinositol-4-phosphate 5-Kinase: M1-L354 HMMER_PFAMPhosphatidylinositol phosphate kinases: M62-T355 HMMER_SMART KINASEPHOSPHATIDYLINOSITOL-4-PHOSPHATE 5-KINASE-TYPE TRANSFERASE BLAST_PRODOMDIPHOSPHOINOSITIDE 1-PHOSPHATIDYLINOSITOL-4-PHOSPHATE PTDINSAP-5- KINASEALPHA PD002308:M1-F112 F112-I353 PHOSPHATIDYLINOSITOL; KINASE;BLAST_DOMO DM07197|P48426|8-404:G8-Q113 Q113-T355 PHOSPHATIDYLINOSITOL;KINASE; BLAST_DOMO DM07197|P38994|351-756:L41-F112 Q110-1350 PotentialPhosphorylation Sites: S48, S150, S171, S296, S343, T18, T181, T261,T311, T325 MOTIFS Potential Glycosylation Sites: N46 MOTIFS 9 7516525CD1543 Protein kinase domain: Y128-V447 HMMER_PFAM Serine/Threonine proteinkinases, catalytic domain: Y128-V447 HMMER_SMART Receptor tyrosinekinase class V IPB001426: L294-K315, P316-D342 BLIMPS_BLOCKS Proteinkinases signatures and profile: Q289-D342 PROFILESCAN Tyrosine kinasecatalytic domain signature PR00109: Y303-L321, I416-V438, G350-I360,L372- BLIMPS_PRINTS D394 KINASE TRANSFERASE ATP-BINDINGSERINE/THREONINE-PROTEIN TYROSINE- BLAST_PRODOM PROTEIN RECEPTOR2.7.1.-PHOSPHORYLATION PRECURSOR PD000001:Q127-A353 G340-E453 P414-W446KINASE ATP-BINDING TRANSFERASE SERINE/THREONINE-PROTEIN BLAST_PRODOMCA2/CALMODULIN-DEPENDENT BETA CG17698 CA/CALMODULIN-DEPENDENT ALPHASERINE/THREONINE PD019141: V447-F501 KINASE ATP-BINDINGSERINE/THREONINE-PROTEIN CA2/CALMODULIN- BLAST_PRODOM DEPENDENTTRANSFERASE ALPHA SERINE/THREONINE GLYCOGEN CALCIUM/ CALMODULINPD027014: E502-S543 KINASE ATP-BINDING SERINE/THREONINE-PROTEINTRANSFERASE BLAST_PRODOM CA2/CALMODULIN-DEPENDENT BETACA/CALMODULIN-DEPENDENT ALPHA SERINETHREONINE PD031900: M1-Q127 PROTEINKINASE DOMAIN BLAST_DOMO DM00004|A57156|130-399:L130-L228 Q238-V438PROTEIN KINASE DOMAIN BLAST_DOMO DM00004|P50526|136-399:E133-Q231P247-1436 PROTEIN KINASE DOMAIN BLAST_DOMODM00004|P06782|57-296:I134-K166 R195-Q231 D282-V438 PROTEIN KINASEDOMAIN BLAST_DOMO DM00004|JC1446|20-261:K129-L164 E194-Q231 V267-V438Potential Phosphorylation Sites: S69, S74, S82, S100, S117, S160, S266,S368, S457, S463, MOTIFS S475, S496, T26, T58, T108, T468 PotentialGlycosylation Sites: N147 MOTIFS ATP/GTP-binding site motif A (P-loop):G523-S530 MOTIFS Protein kinases ATP-binding region signature: I134-K157MOTIFS Serine/Threonine protein kinases active-site signature: I309-L321MOTIFS 10 7516533CD1 445 Protein kinase domain: I30-F272 HMMER_PFAMProtein kinase C terminal domain: R273-I359 HMMER_PFAM Extension toSer/Thr-type protein kinases: R273-A335 HMMER_SMART Serine/Threonineprotein kinases, catalytic domain: E41-F272 HMMER_SMART Receptortyrosine kinase class II IPB002011: I66-F110, I134-D185, N217-G261BLIMPS_BLOCKS Tyrosine kinase catalytic domain signature PR00109:H128-L146, V194-E216, L92-E105, L236- BLIMPS_PRINTS A258 KINASE S6RIBOSOMAL SERINE/THREONINE-PROTEIN TRANSFERASE P70 BETA BLAST_PRODOM2.7.1.-ATP-BINDING PHOSPHORYLATION PD032092: S337-L445 PROTEIN KINASEDOMAIN BLAST_DOMO DM00004|A53300|64-305: K55-G257 DM00004|A57459|61-302:V27-G251 DM00004|P23443|69-313: A48-G257 PROTEIN KINASE C ALPHABLAST_DOMO DM04692|A37237|11-676: 150-V334 Potential PhosphorylationSites: S40, S96, S163, S295, S300, S314, S337, S341, S354, S361, MOTIFSS372, S399, S435, T60, T221, T310, T319, T390, Y11 Serine/Threonineprotein kinases active-site signature: I134-L146 MOTIFS 11 7516613CD11219 CNH domain: Y901-R1199 HMMER_PFAM Protein kinase domain: F25-I289HMMER_PFAM Domain found in NIK1-like kinases, mouse citron and yeastROM1, ROM2: Y901-R1199 HMMER_SMART Serine/Threonine protein kinases,catalytic domain: F25-I289 HMMER_SMART Tyrosine kinase, catalyticdomain: F25-I289 HMMER_SMART Receptor tyrosine kinase class IIIIPB001824: T59-I113, W129-K168, G190-P232 BLIMPS_BLOCKS Protein kinasessignatures and profile: W129-V182 PROFILESCAN KINASESERINE/THREONINE-PROTEIN BINDING PHORBOL-ESTER ATP-BINDING BLAST_PRODOMTRANSFERASE GDP-GTP EXCHANGE RHO1 CDC42-BINDING PD014445:L919-S1043F1074-S1197 KINASE SERINE/THREONINE-PROTEIN ATP-BINDING TRANSFERASEMIG-15 BLAST_PRODOM TYROSINE-PROTEIN 2.7.1.- PD147188:I289-P500S795-W915 COIL COILED MYOSIN CHAIN ATP-BINDING HEAVY FILAMENT MUSCLEREPEAT BLAST_PRODOM INTERMEDIATE PD000002: K316-K517, Q292-Q471,Q301-Q490, L352-R569, 1289-E466, Q292-R459, R358-E537 ATP-BINDINGTRANSFERASE NIK KINASE SERINE/THREONINE-PROTEIN PD147187: BLAST_PRODOMH501-K831, E514-W915 PROTEIN KINASE DOMAIN BLAST_DOMODM00004|A53714|17-262: L27-S279 DM00004|P08458|20-262: V31-S279DM00004|P10676|18-272: L27-P278 DM00004|P38692|24-266: E29-S279Potential Phosphorylation Sites: S9, S17, S77, S112, S255, S259, S264,S324, S326, S550, MOTIFS S554, S573, S625, S626, 5633, S682, S683, S707,S721, S727, S756, S764, S880, S963, S1023, S1043, S1083, S1096, S1197,T59, T124, T187, T222, T309, T319, T351, T543, T689, T690, T810, T816,T876, T996, T1057, Y321, Y323, Y467 MOTIFS Potential GlycosylationSites: N33, N570, N719, N818, N1151 MOTIFS Leucine zipper pattern:L472-L493 MOTIFS Protein kinases ATP-binding region signature: V31-K54MOTIFS Serine/Threonine protein kinases active-site signature: V149-L161MOTIFS 12 7517068CD1 1168 CNH domain: Y850-R1148 HMMER_PFAM Proteinkinase domain: F25-I289 HMMER_PFAM Domain found in NIK1-like kinases,mouse citron and yeast ROM1, ROM2: Y850-R1148 HMMER_SMARTSerine/Threonine protein kinases, catalytic domain: F25-I289 HMMER_SMARTTyrosine kinase, catalytic domain: F25-I289 HMMER_SMART Eukaryoticprotein kinase IPB000719: H145-L160, Y210-G220 BLIMPS_BLOCKS Receptortyrosine kinase class III IPB001824: T59-V113, W129-K168, G190-P232BLIMPS_BLOCKS Protein kinases signatures and profile: W129-T181PROFILESCAN Tyrosine kinase catalytic domain signature PR00109:M105-K118, H143-L161, S214-M236, BLIMPS_PRINTS G190-I200, W258-T280KINASE SERINE/THREONINE-PROTEIN BINDING PHORBOL-ESTER ATP-BINDINGBLAST_PRODOM TRANSFERASE GDP-GTP EXCHANGE RHO1 CDC42-BINDINGPD014445:L868-S992 F1023-S1146 KINASE SERINE/THREONINE-PROTEINATP-BINDING TRANSFERASE MIG-15 BLAST_PRODOM TYROSINE-PROTEIN 2.7.1.-PD147188:1289-E648 V831-W864 KINASE SERINE/THREONINE-PROTEIN ATP-BINDINGTRANSFERASE NCK TRAF2 BLAST_PRODOM INTERACTING VARIANT SPLICE GCKPD043898: F993-P1039 ATP-BINDING TRANSFERASE NIK KINASESERINE/THREONINE-PROTEIN PD147187: BLAS_PRODOM R402-G756, S545-W864PROTEIN KINASE DOMAIN BLAST_DOMO DM00004|A53714|17-262: L27-P278DM00004|P10676|18-272: L27-P278 DM00004|P38692|24-266: E29-R277DM00004|P50527|388-627: V31-T280 Potential Phosphorylation Sites: S9,S77, S112, S255, S259, S264, S275, S324, S326, S426, MOTIFS S446, S504,S523, S571, S580, S639, S640, S646, S647, S696, S723, S767, S776, S793,S829, S912, S992, S1146, T59, T124, T187, T222, T309, T319, T349, T467,T627, T635, T716, T750, T795, T945, T1006, Y321, Y323 PotentialGlycosylation Sites: N33, N273, N333, N443, N507 MOTIFS Protein kinasesATP-binding region signature: V31-K54 MOTIFS Serine/Threonine proteinkinases active-site signature: V149-L161 MOTIFS 13 7517148CD1 650Regulator of G protein signaling domain: T54-C175 HMMER_PFAM Proteinkinase domain: F191-F453 HMMER_PFAM Regulator of G protein signallingdomain: T54-C175 HMMER_SMART Extension to Ser/Thr-type protein kinases:K454-T533 HMMER_SMART Serine/Threonine protein kinases, catalyticdomain: F191-F453 HMMER_SMART Receptor tyrosine kinase class IIIPB002011: L245-F289, V313-K364, D398-G442 BLIMPS_BLOCKS Tyrosine kinasecatalytic domain signature PR00109: L271-S284, H307-L325, F417-C439BLIMPS_PRINTS GPCR kinase signature PR00717: F171-N183, K230-T248,P468-I485, T493-Y506, K507-T524 BLIMPS_PRINTS Regulator of G proteinsignalling domain proteins PF00615: M15-K21, F162-K178, I270-L283BLIMPS_PFAM PH (pleckstrin homology) domain proteins (P < 0.025)PF00169: S41-L47 BLIMPS_PFAM KINASE RECEPTOR ATP-BINDINGSERINE/THREONINE-PROTEIN TRANSFERASE BLAST_PRODOM COUPLEDBETA-ADRENERGIC MULTI-GENE FAMILY G-PROTEIN PD007430: M1-I53BETA-ADRENERGIC RECEPTOR KINASE COUPLED TRANSFERASE SERINE/ BLAST_PRODOMTHREONINE PROTEIN ATP-BINDING MULTI-GENE FAMILY BETA ARK1PD007640:T533-Q575 BETA-ADRENERGIC RECEPTOR KINASE BETA ARK2 G-PROTEIN COUPLEDBLAST_PRODOM TRANSFERASE SERINE/THREONINE PROTEIN ATP-BINDING MULTI-GENEPD151831: T612-L650 PROTEIN KINASE DOMAIN BLAST_DOMODM00004|P21146|193-437: V193-G438 DM00004|P32865|193-438: V193-0438DM00004|Q09537|205-450: V193-C439 DM00004|Q09639|193-439: V193-G438Potential Phosphorylation Sites: S29, S38, S137, S156, S168, S247, S290,S343, S370, S423, MOTIFS S434, S487, S514, S596, S598, T187, T213, T366,T524, T533, T612, Y92 Potential Glycosylation Sites: N610 MOTIFS Proteinkinases ATP-binding region signature: I197-K220 MOTIFS Serine/Threonineprotein kinases active-site signature: V313-L325 MOTIFS 14 7517238CD1603 Kinase associated domain 1: 5554-V603 HMMER_PFAM Protein kinasedomain: Y11-I215 HMMER_PFAM Serine/Threonine protein kinases, catalyticdomain: Y11-1215 HMMER_SMART KINASE SERINE/THREONINE-PROTEIN ATP-BINDINGTRANSFERASE ZIPPER BLAST_PRODOM MATERNAL EMBRYONIC LEUCINE PK38 W03G1.6PD017644: I215-V603 PROTEIN KINASE DOMAIN BLAST_DOMODM00004|S52244|15-255:L13-E87 E88-M206 PROTEIN KINASE DOMAIN BLAST_DOMODM00004|P06782|57-296:E15-D93 E88-M206 PROTEIN KINASE DOMAIN BLAST_DOMODM00004|P54645|17-258:L13-E87 E88-M206 PROTEIN KINASE DOMAIN BLAST_DOMODM00004|S51025|18-258:L13-E87 E88-M206 Potential Phosphorylation Sites:S140, S205, S308, S315, S496, S501, S600, T56, T252, T313, MOTIFS T339,T380, T439, T441, T470, T517, T547, T552, Y10, Y379, Y590 PotentialGlycosylation Sites: N306, N437, N514 MOTIFS Leucine zipper pattern:L117-L138 MOTIFS Protein kinases ATP-binding region signature: I17-K40MOTIFS 15 7518685CD1 750 Protein-tyrosine phosphatase: N54-E231HMMER_PFAM Protein tyrosine phosphatase, catalytic domain: E23-K234HMMER_SMART Protein tyrosine phosphatase, catalytic domain motif:T127-E231 HMMER_SMART Tyrosine specific protein phosphatase and dualspecificity protein phosphatase family BLIMPS_BLOCKS IPB000387:I168-G178 Tyrosine specific protein phosphatases signature and profiles:P147-V239 PROFILESCAN Protein tyrosine phosphatase signature PR00700:S83-I90, Y99-E119, R126-S143, P165-I183, BLIMPS_PRINTS F199-S214,L215-V225 Salmonella/Yersinia modular tyrosine phosphatase signaturePR01371: E124-D138, I166-T177 BLIMPS_PRINTS HYDROLASE PHOSPHATASEPROTEIN PROTEIN TYROSINE PRECURSOR SIGNAL BLAST_PRODOM TYROSINETRANSMEMBRANE GLYCOPROTEIN RECEPTOR PD000167:N54-E119 F131- E231HYDROLASE PHOSPHATASE PROTEIN PROTEIN TYROSINE TYROSINE PRECURSORBLAST_PRODOM SIGNAL TRANSMEMBRANE GLYCOPROTEIN RECEPTOR PD000155:E124-Q236 HEMATOPOIETIC CELL PROTEIN TYROSINE PHOSPHATASE 70ZPEPHYDROLASE BLAST_PRODOM PD143889: E223-1750 HEMATOPOIETIC CELL PROTEINTYROSINE PHOSPHATASE 70ZPEP HYDROLASE BLAST_PRODOM PD166993: M1-K53PROTEIN-TYROSINE-PHOSPHATASE BLAST_DOMO DM00089|P29352|22-291:E22-L123E124-R235 PROTEIN-TYROSINE-PHOSPHATASE BLAS_DOMODM00089|I48666|14-296:K21-L123 E124-Q236 PROTEIN-TYROSINE-PHOSPHATASEBLAST_DOMO DM00089|Q05209|14-295:K21-L123 E124-R235PROTEIN-TYROSINE-PHOSPHATASE BLAST_DOMO DM00089|S48748|14-295:K21-L123E124-R235 Potential Phosphorylation Sites: S16, S35, S69, S78, S121,S143, S245, S295, S305, S413, MOTIFS S436, S489, S619, S624, S667, S677,S694, S736, T20, T47, T77, T109, T210, T275, T287, T319, T337, T393,T595, T681, Y44, Y66 Potential Glycosylation Sites: N198, N259, N327,N411, N441, N454, N534, N674, N721, N722 MOTIFS Tyrosine specificprotein phosphatases active site: I168-I180 MOTIFS 16 7520192CD1 206signal_cleavage: M1-A42 SPSCAN FERM domain (Band 4.1 family): C31-H149HMMER_PFAM Band 4.1 homologues: Q25-H149 HMMER_SMART Band 4.1 familyIPB000299: E129-K172 BLIMPS_BLOCKS Band 4.1 family domain signatures andprofile: K89-E131 PROFILESCAN Band 4.1 family domain signatures andprofile: G124-K172 PROFILESCAN Band 4.1 protein family signaturePR00935: L62-L74, E129-G145 BLIMPS_PRINTS PROTEIN CYTOSKELETONSTRUCTURAL PHOSPHATASE HYDROLASE PROTEIN BLAST_PRODOM TYROSINEPHOSPHORYLATION MOESIN TYROSINE BAND PD000961:V30-R123 R123- F148PROTEIN CYTOSKELETON STRUCTURAL PROTEIN TYROSINE PHOSPHATASEBLAST_PRODOM HYDROLASE BAND ALTERNATIVE SPLICING PHOSPHORYLATIONPD014063: H149- E202 PROTEIN TYROSINE PHOSPHATASE MEG1 EC 3.1.3.48PTPASE MEG1 MEG BLAST_PRODOM STRUCTURAL PROTEIN CYTOSKELETON HYDROLASEPD129232: M1-V30 BAND 4 BLAST_DOMO DM00609|P29074|19-463:E19-R123F110-L203 BAND 4 BLAST_DOMO DM00609|P11171|200-623:R24-R123 K116-R200BAND 4 BLAST_DOMO DM00609|P11434|183-612:R24-R123 K116-R200 BAND 4BLAST_DOMO DM00609|P28191|18-438:E28-R123 G124-E202 PotentialPhosphorylation Sites: S99, S126, S155, S189, T2, T65, T137 MOTIFS Band41 family domain signature 1: W84-D113 MOTIFS 17 7520428CD1 733 SignalPeptide: M31-A54 HMMER signal_cleavage: M1-S68 SPSCAN Protein kinasedomain: F427-F700 HMMER_PFAM Serine/Threonine protein kinases, catalyticdomain: F427-F700 HMMER_SMART Eukaryotic protein kinase IPB000719:H542-L557, Y617-G627 BLIMPS_BLOCKS Protein kinases signatures andprofile: F494-M574 PROFILESCAN Tyrosine kinase catalytic domainsignature PR00109: M504-K517, Y540-I558, V621-D643 BLIMPS_PRINTS PROTEINKINASE SERINE/THREONINE KIN4 MICROTUBULE ASSOCIATED TESTIS BLAST_PRODOMSPECIFIC TESTIS-SPECIFIC MAST205 PD041650: K236-D426 MICROTUBULEASSOCIATED TESTIS SPECIFIC SERINE/THREONINE PROTEIN BLAST_PRODOM KINASE205 KD TESTIS-SPECIFIC SERINE/THREONINE PROTEIN KINASE MAST205 KINASEPD135564: C83-Y235 PROTEIN KINASE DOMAIN BLAST_DOMODM08046|P05986|1-397:D183-P206 S423-K573 V600-E733 PROTEIN KINASE DOMAINBLAST_DOMO DM00004|S42867|75-498:I430-T581 H587-F728 PROTEIN KINASEDOMAIN BLAST_DOMO DM00004|S42864|41-325:E428-K573 H587-T688 PROTEINKINASE DOMAIN BLAST_DOMO DM00004|A54602|455-712: T429-G687 PotentialPhosphorylation Sites: S75, S82, S86, S115, S119, S145, S168, S196,S395, S418, MOTIFS S423, S448, S690, S721, S726, T181, T421, T429, T480,T496, T644, T674, T701, T730 Leucine zipper pattern: L515-L536 MOTIFSSerine/Threonine protein kinases active-site signature: I546-I558 MOTIFS18 7522586CD1 114 PITSLRE ALPHA ISOFORM PROTEIN KINASE PBETA22 CELLDIVISION CYCLE 2LIKE BLAST_PRODOM PD009467: M1-K108 PotentialPhosphorylation Sites: S7, S43, S47, S72, S92, T12, T61 MOTIFS 197524017CD1 612 Kinase associated domain 1: S563-V612 HMMER_PFAM Proteinkinase domain: Y11-I224 HMMER_PFAM Serine/Threonine protein kinases,catalytic domain: Y11-I224 HMMER_SMART Receptor tyrosine kinase class VIPB001426: F74-K95, P96-K122, C129-Y161 BLIMPS_BLOCKS Phorbolesters/diacylglycerol binding domain IPB002219: T16-K26, V84-D93,C130-E139 BLIMPS_BLOCKS Tyrosine kinase catalytic domain signaturePR00109: L47-I60, Y83-F101, A151-D173, L193- BLIMPS_PRINTS M215 KINASEPROTEIN KIAA0175 PK38 MATERNAL EMBRYONIC LEUCINE ZIPPER BLAST_PRODOMSERINE/THREONINE P69EG3 PD017644: I224-V612 PROTEIN KINASE DOMAINBLAST_DOMO DM00004|S52244|15-255:L13-G48 L47-M215 PROTEIN KINASE DOMAINBLAST_DOMO DM00004|P06782|57-296:E15-L47 G52-M215 PROTEIN KINASE DOMAINBLAST_DOMO DM00004|P54645|17-258:L13-L47 Y49-M215 PROTEIN KINASE DOMAINBLAST_DOMO DM00004|S24578|18-262:L13-L47 Y49-M215 PotentialPhosphorylation Sites: S66, S149, S214, S317, S324, S505, S510, S609,T261, T322, MOTIFS T348, T389, T448, T450, T479, T526, T556, T561, Y10,Y388, Y599 Potential Glycosylation Sites: N315, N446, N523 MOTIFSLeucine zipper pattern: L126-L147 MOTIFS Prenyl group binding site (CAAXbox): MOTIFS Protein kinases ATP-binding region signature: I17-K40MOTIFS Serine/Threonine protein kinases active-site signature: Y89-F101MOTIFS 20 7525773CD1 311 GHMP kinases putative ATP-binding protein:V5-P311 HMMER_PFAM mevalonate kinase: L7-S309 HMMER_PFAM GHMP kinasesputative ATP-binding domain IPB001745: P11-H20, H276-C287 BLIMPS_BLOCKSMevalonate kinase signature PR00959: A10-N34, E141-G160, H276-K293BLIMPS_PRINTS KINASE ATP-BINDING TRANSFERASE GALACTOKINASE GALACTOSEBLAST_PRODOM METABOLISM MEVALONATE MK BIOSYNTHESIS PROTEIN PD002375:I144-L292 MEVALONATE KINASE TRANSFERASE ATP-BINDING MK BIOSYNTHESISSTEROL BLAST_PRODOM PROTEIN CHOLESTEROL MVK PD007691: L6-I58 MEVALONATEKINASE MK TRANSFERASE CHOLESTEROL BIOSYNTHESIS ATP- BLAST_PRODOM BINDINGDISEASE MUTATION PD013931: K59-R124 GHMP KINASES PUTATIVE ATP-BINDINGDOMAIN BLAST_DOMO DM02935|Q03426|1-379:M1-R124 R124-I296 GHMP KINASESPUTATIVE ATP-BINDING DOMAIN BLAST_DOMO DM02935|S42226|162-540:M1-R124R124-I296 GHMP KINASES PUTATIVE ATP-BINDING DOMAIN BLAST_DOMODM02935|P46086|1-376:A10-I119 A95-G295 GHMP KINASES PUTATIVE ATP-BINDINGDOMAIN BLAST_DOMO DM02935|Q09780|1-373:L2-I119 L130-P294 PotentialPhosphorylation Sites: S73, S149, S171, S302, T104, T126, T214 MOTIFS 217525861CD1 206 Protein tyrosine phosphatase signature PR00700:F165-G180, M181-C191 BLIMPS_PRINTS PROTEIN TYROSINE PHOSPHATASE,NON-RECEPTOR TYPE 20 EC 3.1.3.48 PHOSPHO- BLAST_PRODOM TYROSINEPHOSPHATASE PTPASE HYDROLASE PD097276: M1-M164 Potential PhosphorylationSites: S3, S20, S34, S51, S57, S76, S95, S96, S120, T39, T84, T115MOTIFS Potential Glycosylation Sites: N18 MOTIFS 22 2509577CD1 1125Armadillo/beta-catenin-like repeat: V198-E238, S239-A279, E280-E320,N401-S448 HMMER_PFAM Protein kinase domain: Y519-I783 HMMER_PFAMArmadillo/beta-catenin-like repeats:A197-E238, C278-E320, N401-S448HMMER_SMART Serine/Threonine protein kinases, catalyti: Y519-L791HMMER_SMART Tyrosine kinase, catalytic domain: Y519-I783 HMMER_SMARTReceptor tyrosine kinase class II IPB002011: I578-K622, I651-K702,L735-V779 BLIMPS_BLOCKS Protein kinases signatures and profile:R630-S684 PROFILESCAN Tyrosine kinase catalytic domain signaturePR00109: L645-L663, A710-T732, Y754-I776 BLIMPS_PRINTS SERINE/THREONINEPROTEIN KINASE D1044.3 IN CHROMOSOME III EC 2.7.1. BLAST_PRODOM PROTEINTRANSFERASE ATP-BINDING EGF-LIKE DOMAIN PD140750:E108-I334 D775-Q813C319-G517 PROTEIN KINASE DOMAIN DM00004 BLAST_DOMO|P11837|13-285:I521-H646 I651-D775 |P41951|433-687:I521-I776|P51954|6-248: L522-I776 |P51955|10-261: I521-I776 PotentialPhosphorylation Sites: S40, S235, S266, S376, S479, S540, S567, S618,S696, S755, MOTIFS S852, S900, S935, S974, S1039, S1068, S1108, T11,T15, T22, T58, T270, T324, T593, T670, T812, T898, T1077, T1092, T1123,Y746 Potential Glycosylation Sites: N86, N96, N187, N401, N793, N911,N1105 MOTIFS Protein kinases ATP-binding region signature: L525-K548MOTIFS Tyrosine protein kinases specific active-site signature:I651-L663 MOTIFS 23 7505222CD1 888 Protein kinase domain: Y61-L316HMMER_PFAM Serine/Threonine protein kinases, catalyti: Y61-L316HMMER_SMART Tyrosine kinase, catalytic domain: Y61-L316 HMMER_SMARTReceptor tyrosine kinase class III IPB001824: M97-R151, Q161-A200,A218-P260 BLIMPS_BLOCKS Protein kinases signatures and profile:Q161-S214 PROFILESCAN Tyrosine kinase catalytic domain signaturePR00109: M137-N150, H175-L193, T242-N264, BLIMPS_PRINTS F285-I307PROTEIN KINASE DOMAIN DM00004 BLAST_DOMO |P51954|6-248: I64-I307|P51955|10-261: V63-I307 |P51957|8-251: I64-I307 |Q08942|22-269:I67-I307 Potential Phosphorylation Sites: S53, S105, S126, S300, S399,S487, S501, S556, S574, S754, MOTIFS S781, S794, S804, S838, T32, T256,T442, T640, T660, T711, T763, T799, T821, T834, T865, T873, Y474, Y605Potential Glycosylation Sites: N212, N240, N636, N861 MOTIFS Proteinkinases ATP-binding region signature: I67-K90 MOTIFS Serine/Threonineprotein kinases active-site signature: I181-L193 MOTIFS 24 7524408CD1487 GDA1/CD39 (nucleoside phosphatase) family: T80-K487 HMMER_PFAMCytosolic domain: M1-I34 TMHMMER Transmembrane domain: M35-I54Non-cytosolic domain: R55-K487 GDA1/CD39 family of nucleosidephosphatase IPB000407: I91-Y105, P173-R183, I217-E238, BLIMPS_BLOCKSG268-Y281 HYDROLASE TRANSMEMBRANE PROTEIN NUCLEOSIDE CD39 NUCLEOSIDE-BLAST_PRODOM TRIPHOSPHATASE TRIPHOSPHATE NTP-ASE PRECURSOR ATP-DIPHOSPHOHYDROLASE PD003822:N86-K487 I91-Y105 GUANOSINEDIPHOSPHATASE-LIKE PROTEIN KIAA0392 PD070805: M1-P85 BLAST_PRODOMACTIVATION; NUCLEOSIDE; ANTIGEN; LYMPHOID; DM02628 BLAST_DOMO|I56242|40-471:N86-S291 K264-F472 |P49961|40-471:N86-S291 K264-F472|P32621|84-517:Y89-G235 T266-Y435 |P40009|1-462: N84-S479 PotentialPhosphorylation Sites: S212, S218, S292, S479, T75, T144, T266, Y175,Y477 MOTIFS Potential Glycosylation Sites: N404, N407 MOTIFS 257526163CD1 1309 PDZ domain (Also known as DHR or GLGF): P950-L1037HMMER_PFAM Protein kinase domain: F367-F640 HMMER_PFAM Domain present inPSD-95, Dlg, and ZO-1/2: K958-E1038 HMMER_SMART Extension toSer/Thr-type protein kinases: L641-F704 HMMER_SMART Serine/Threonineprotein kinases, catalyt: F367-F640 HMMER_SMART Eukaryotic proteinkinase IPB000719: H482-L497, Y557-G567 BLIMPS_BLOCKS Tyrosine kinasecatalytic domain signature PR00109: M444-K457, Y480-1498, V561-D583BLIMPS_PRINTS PROTEIN KINASE SERINE/THREONINE KIN4 MICROTUBULEASSOCIATED TESTIS BLAST_PRODOM SPECIFIC TESTIS SPECIFIC MAST205PD041650: R177-D366 MICROTUBULE ASSOCIATED TESTIS SPECIFICSERINE/THREONINE PROTEIN BLAST_PRODOM KINASE 205 KD TESTIS SPECIFICSERINE/THREONINE PROTEIN KINASE MAST205 KINASE PD069998: T1034-D1128MICROTUBULE ASSOCIATED TESTIS SPECIFIC SERINE/THREONINE PROTEINBLAST_PRODOM KINASE 205 KD TESTIS SPECIFIC SERINE/THREONINE PROTEINKINASE MAST205 KINASE PD135564: G11-Y176 MICROTUBULE ASSOCIATED TESTISSPECIFIC SERINE/THREONINE PROTEIN BLAST_PRODOM KINASE 205 KD TESTISSPECIFIC SERINE/THREONINE PROTEIN KINASE MAST205 KINASE PD182663:T719-W984 PROTEIN KINASE DOMAIN BLAST_DOMO DM00004|S42867|75-498:I370-K513 H527-F668 A1135-A1148 |A54602|455-712:T369-G627 PROTEIN KINASE DOMAIN BLAST_DOMO DM08046|P05986|1-397:S365-K513 V540-E684 |P06244|1-396:D366-K513 V540-E684Potential Phosphorylation Sites: S57, S61, S85, S108, S146, S257, S336,S358, S365, S621, MOTIFS S666, S672, S680, S690, S701, S709, S726, S738,S747, S767, S792, S793, S921, S930, S946, S956, S963, S985, S1031,S1041, S1060, S1063, S1074, S1080, S1101, S1180, S1273, S1215, S1257,S1262, T1068, T1181, T121, T420, T584, T670, T685, T721, T907, T1035,T1036, T235, T369, T436, T854, T1088, T1175, T1036 PotentialGlycosylation Sites: N64, N1039 MOTIFS Serine/Threonine protein kinasesactive-site signature: I486-I498 MOTIFS 26 7526158CD1 1331 PDZ domain(Also known as DHR or GLGF): P972-L1059 HMMER_PFAM Protein kinasedomain: F389-F662 HMMER_PFAM Domain present in PSD-95, Dlg, and ZO-1/2:K980-E1060 HMMER_SMART Serine/Threonine protein kinases, catalyt:F389-F662 HMMER_SMART Eukaryotic protein kinase IPB000719: H504-L519,Y579-G589 BLIMPS_BLOCKS Tyrosine kinase catalytic domain signaturePR00109: M466-K479, Y502-I520, V583-D605 BLIMPS_PRINTS PROTEIN KINASESERINE/THREONINE IGN4 MICROTUBULE ASSOCIATED TESTIS BLAST_PRODOMSPECIFIC TESTIS SPECIFIC MAST205 PD041650: R199-D388 MICROTUBULEASSOCIATED TESTIS SPECIFIC SERINE/THREONINE PROTEIN BLAST_PRODOM KINASE205 KD TESTIS SPECIFIC SERINE/THREONINE PROTEIN KINASE MAST205 KINASEPD069998: T1056-D1150 MICROTUBULE ASSOCIATED TESTIS SPECIFICSERINE/THREONINE PROTEIN BLAST_PRODOM KINASE 205 KD TESTIS SPECIFICSERINE/THREONINE PROTEIN KINASE MAST205 KINASE PD135564: C47-Y198MICROTUBULE ASSOCIATED TESTIS SPECIFIC SERINE/THREONINE PROTEINBLAST_PRODOM KINASE 205 KD TESTIS SPECIFIC SERINE/THREONINE PROTEINKINASE MAST205 KINASE PD182663: T741-W1006 PROTEIN KINASE DOMAINBLAST_DOMO DM00004 |S42867|75-498:I392-K535 H549-F690 A1157-A1170|A54602|455-712: T391-G649 PROTEIN KINASE DOMAIN DM08046 BLAST_DOMO|P05986|1-397:S387-K535 V562-E706 |P06244|1-396:D388-K535 V562-E706Potential Phosphorylation Sites: S3, S46, S79, S83, S107, S130, S168,S279, S358, S380, S387, MOTIFS S643, S688, S694, S702, S712, S723, S731,S748, S760, S769, S789, S814, S815, S943, S952, S968, S978, S985, S1007,S1053; S1063, S1082, S1085, S1096, S1102, S1123, S1202, S1284, S1295,S1237, S1279, S1284, T1090, T1203, T143, T442, T606, T692, T707, T743,T929, T1057, T1328, T257, T391, T458, T876, T1110, T1197, T1328Serine/Threonine protein kinases active-site signature: I508-I520 MOTIFS27 7519807CD1 80 MAM domain IPB000998: C54-V66 BLIMPS_BLOCKS MAM domainsignature PR00020: G52-K70 BLIMPS_PRINTS Potential PhosphorylationSites: T27, T34 MOTIFS 28 7526180CD1 495 ACIDIC SERINE CLUSTER REPEATBLAST_DOMO DM03496|P32583|57-405: S261-D495 Potential PhosphorylationSites: S141, S217, S239, S294, S296, S359, S430, S442, S451, S466,MOTIFS S476, T194, T196, T241, T251, T342, T375, T392, T412, T417Potential Glycosylation Sites: N192, N220, N289, N465 MOTIFS 297526185CD1 157 Serine/Threonine protein kinases, catalytic domain:F24-Y157 HMMER_SMART KINASE PROTEIN DOMAIN TRANSFERASE PD00584: L27-G36BLIMPS_(—) PRODOM PROTEIN KINASE DOMAIN BLAST_DOMODM00004|A53714|17-262: L27-V151 DM00004|I49376|270-509: K26-G153DM00004|P08458|20-262: I30-V151 DM00004|P38692|24-266: K26-V151Potential Phosphorylation Sites: S34, S75, S106, S137, T25, T46 MOTIFSPotential Glycosylation Sites: N44 MOTIFS Protein kinases ATP-bindingregion signature: I30-K53 MOTIFS 30 7526192CD1 305 Protein kinasedomain: F46-G305 HMMER_PFAM Serine/Threonine protein kinases, catalyticdomain: F46-G305 HMMER_SMART Eukaryotic protein kinase IPB000719:H189-L204 BLIMPS_BLOCKS Protein kinases signatures and profile:T173-P230 PROFILESCAN CASEIN KINASE I GAMMA ISOFORM CKIGAMMA TRANSFERASEBLAST_PRODOM SERINE/THREONINEPROTEIN ATPBINDING MULTIGENE PD026544:M1-N45 PROTEIN KINASE DOMAIN BLAST_DOMO DM00004|B56711|48-303:V48-L76E109-R302 DM00004|A56711|46-303:V48-L76 E109-R302DM00004|C56711|45-301:V48-L76 E109-R302 DM00004|D56406|31-276:V48-L76E109-R302 Potential Phosphorylation Sites: S19, S99, S129, S262, T84,T183, T210, T232, T247 MOTIFS Protein kinases ATP-binding regionsignature: I52-K75 MOTIFS Serine/Threonine protein kinases active-sitesignature: L193-V205 MOTIFS 31 7526193CD1 930 Signal cleavage: M1-G68SPSCAN Protein kinase domain: V46-F310 HMMER_PFAM Serine/Threonineprotein kinases, catalytic domain: V46-K313 HMMER_SMART Eukaryoticprotein kinase IPB000719: C168-L183, I239-G249 BLIMPS_BLOCKS PROTEINREPEAT SIGNAL PRECURSOR PRION GLYCOPROTEIN NUCLEAR BLAST_PRODOMGPIANCHOR BRAIN MAJOR PD001091: G373-P626, G404-P626, P358-Q601,P349-Q574, P320-S519, P296-Q541 PROTEIN KINASE DOMAIN BLAST_DOMODM00004|P38080|36-309: L52-I304 DM00004|P40494|23-287: L52-I304DM00004|P51954|6-248: L52-I304 DM00004|P53974|23-288: L52-I304 PotentialPhosphorylation Sites: S7, S115, S224, S235, S311, S625, S679, S785,S815, S822, MOTIFS S833, S871, S879, T47, T147, T199, T221, T240, T241,T275, T389, T395, T628, T708, T743, T757, T829 Potential GlycosylationSites: N113, N273, N667, N703, N823, N905 MOTIFS Serine/Threonineprotein kinases active-site signature: I172-L184 MOTIFS 32 7526196CD1118 Signal Peptide: M1-G22 HMMER Signal_cleavage: M1-G22 SPSCANSerine/threonine dehydratase pyridoxal-phosphate attachment siteIPB000634: E95-S104 BLIMPS_BLOCKS CYCLIN G-ASSOCIATED KINASE TRANSFERASESERINE/THREONINEPROTEIN BLAST_PRODOM ATPBINDING HSGAK PD026473: M1-L40Potential Phosphorylation Sites: S6, S21, S62, S73, S92, S113 MOTIFS 337526198CD1 1355 Protein kinase domain: L40-E315 HMMER_PFAM DnaJmolecular chaperone homology domain: E1290-S1351 HMMR_SMARTSerine/Threonine protein kinases, catalytic domain: L40-A317 HMMER_SMARTEukaryotic protein kinase IPB000719: Q165-L180, I240-G250 BLIMPS_BLOCKSProtein kinases signatures and profile: V148-H200 PROFILESCAN CYCLING-ASSOCIATED KINASE TRANSFERASE SERINE/THREONINEPROTEIN BLAST_PRODOMATPBINDING HSGAK PD039449: A317-N402 PROTEIN AUXILIN COAT REPEATPHOSPHORYLATION KIAA0473 CYCLIN G- BLAST_PRODOM ASSOCIATED KINASETRANSFERASE PD010124: Q1215-Q1349 PD025411: S456-V640 PD151518:L641-L1093, P868-S1235, R320-E366 PROTEIN KINASE DOMAIN BLAST_DOMODM00004|P38080|36-309: L46-I306 DM00004|P40494|23-287: R41-I306DM00004|P53974|23-288: R44-I306 DM00004|Q09170|169-423: R44-S305Potential Phosphorylation Sites: S6, S21, S62, S73, S93, S305, S393,S456, S530, S540, S551, MOTIFS S661, S726, S737, S738, S784, S811, S906,S976, S1029, S1103, S1113, S1220, S1234, S1235, S1237, S1344, T155,T186, T382, T414, T459, T611, T680, T776, T805, T949, T1118, T1156,T1165, T1244, Y412 Potential Glycosylation Sites: N677, N724, N809,N970, N1196 MOTIFS Serine/Threonine protein kinases active-sitesignature: I169-L181 MOTIFS 34 7526208CD1 490 Protein kinase domain:Y14-I252 HMMER_PFAM Serine/Threonine protein kinases, catalytic domain:Y14-I252 HMMER_SMART Eukaryotic protein kinase IPB000719: H108-L123,Y171-G181 BLIMPS_BLOCKS Protein kinases signatures and profile: F65-D147PROFILESCAN Tyrosine kinase catalytic domain signature PR00109:H106-L124, V175-E197, V221-A243 BLIMPS_PRINTS KINASE PROTEIN IICALCIUM/CALMODULIN-DEPENDENT TYPE SUBUNIT BLAST_PRODOM CALMODULINBINDINGCHAIN TRANSFERASE SERINE/THREONINEPROTEIN PD001779:1252-K303 S312-V380KINASE PROTEIN II CALCIUM/CALMODULIN-DEPENDENT TYPE SUBUNIT CHAINBLAST_PRODOM TRANSFERASE SERINE/THREONINEPROTEIN CALMODULINBINDINGPD004250: E381-K469 PROTEIN KINASE DOMAIN BLAST_DOMODM00004|JU0270|16-262:E18-R53 V54-A243 DM00004|A44412|16-262:E18-R53V54-A243 DM00004|P11798|15-261: E39-A243, L16-E63 KINASE; DEPENDENT; II;CALMODULIN; BLAST_DOMO DM05068|P11798|263-426: S244-A418 PotentialPhosphorylation Sites: S51, S59, S89, S312, S313, S397, T36, T47, T74,T242, T327, MOTIFS T328, T369 Potential Glycosylation Sites: N293, N326,N479 MOTIFS Protein kinases ATP-binding region signature: L20-K43 MOTIFSSerine/Threonine protein kinases active-site signature: V112-L124 MOTIFS35 7526212CD1 344 Protein kinase domain: Y14-I252 HMMER_PFAMSerine/Threonine protein kinases, catalytic domain: Y14-I252 HMMER_SMARTEukaryotic protein kinase IPB000719: H108-L123, Y171-G181 BLIMPS_BLOCKSProtein kinases signatures and profile: F65-D147 PROFILESCAN Tyrosinekinase catalytic domain signature PR00109: H106-L124, V175-E197,V221-A243 BLIMPS_PRINTS KINASE PROTEIN II CALCIUM/CALMODULIN-DEPENDENTTYPE SUBUNIT BLAST_PRODOM CALMODULINBINDING CHAIN TRANSFERASESERINE/THREONINEPROTEIN PD001779: I252-K324 PROTEIN KINASE DOMAINBLAST_DOMO DM00004|JU0270|16-262:E18-R53 V54-A243DM00004|A44412|16-262:E18-R53 V54-A243 DM00004|P08414|44-285: E19-T242DM00004|P11798|15-261: E39-A243, L16-E63 Potential PhosphorylationSites: S51, S59, S89, T36, T47, T74, T242, T316, T317 MOTIFS PotentialGlycosylation Sites: N293, N315 MOTIFS Protein kinases ATP-bindingregion signature: L20-K43 MOTIFS Serine/Threonine protein kinasesactive-site signature: V112-L124 MOTIFS 36 7526213CD1 89 PotentialPhosphorylation Sites: S5, S56, S80, T52 MOTIFS Hexokinase familyIPB001312: S10-G24 BLIMPS_BLOCKS 37 7526214CD1 88 PotentialPhosphorylation Sites: S5, S56, S67, T52 MOTIFS Hexokinase familyIPB001312: S10-G24 BLIMPS_BLOCKS 38 7526228CD1 137 Signal_cleavage:M1-A15 SPSCAN PROTEIN KINASE DOMAIN BLAST_DOMO DM00004|I38044↑100-349:V38-A117 DM00004|P08630|329-573: E35-N114 DM00004|Q08881|361-604:E35-L112 Potential Phosphorylation Sites: S14, S67, S69 MOTIFS Leucinezipper pattern: L112-L133 MOTIFS Protein kinases ATP-binding regionsignature: V42-K63 MOTIFS 39 7526246CD1 243 Regulator of G proteinsignaling domain: T54-C175 HMMER_PFAM Regulator of G protein signallingdomain: T54-C175 HMMER_SMART GPCR kinase signature PR00717: F171-N183BLIMPS_PRINTS Regulator of G protein signalling domain proteins PF00615:M15-K21, F162-K178 BLIMPS_PFAM RECEPTOR KINASE TRANSFERASESERINE/THREONINEPROTEIN ATPBINDING BLAST_PRODOM BETAADRENERGIC COUPLEDPROTEIN MULTIGENE FAMILY PD007430: M1-V53 KINASE; THREONINE; ATP;SERINE; BLAST_DOMO DM01747|P21146|152-191: E152-S187 N-TERMINAL DOMAINBLAST_DOMO DM05135|P21146|33-150: L33-E151 DM05135|P32865|33-150:L34-E151 DM05135|Q09639|34-149: L34-1150 Potential PhosphorylationSites: S29, S38, S60, S127, S168, T97 MOTIFS Cell attachment sequence:R158-D160 MOTIFS 40 7526258CD1 463 CELL CYCLE PROGRESSION PROTEIN FASTKINASE PD041692: L200-P417 BLAST_PRODOM FAST KINASE PD135789: M1-R201BLAST_PRODOM Potential Phosphorylation Sites: S94, S246, S332, S373,S441, T138, T336, T365 MOTIFS 41 7526311CD1 184 Signal Peptide: M1-G18,M1-A21 HMMER Signal_cleavage: M1-A21 SPSCAN Cytosolic domain: K163-T184TMHMMER Transmembrane domain: WI43-W162 Non-cytosolic domain: M1-T142KINASE DEHYDROGENASE TRANSFERASE PD01976: P54-G66, N69-S117 BLIMPS_(—)PRODOM KINASE PYRUVATE DEHYDROGENASE TRANSFERASE DEHYDROGENASE-BLAST_PRODOM LIPOAMIDE MITOCHONDRIAL PRECURSOR TRANSIT PEPTIDEMITOCHONDRION PD004994: V42-I135 PYRUVATE DEHYDROGENASE-LIPOAMIDE KINASEISOZYME 1, MITOCHONDRIAL BLAST_PRODOM PRECURSOR EC 2.7.1.99DEHYDROGENASE ISOFORM 1 TRANSFERASE TRANSIT PEPTIDE MITOCHONDRIONMULTIGENE FAMILY PD174825: M1-E39 KINASE; DEHYDROGENASE; PYRUVATE; ACID;BLAST_DOMO DM01978|A55305|2-103: A37-E130 DM01978|I55465|28-129:F28-E130 DM01978|I70159|2-103: A37-E130 DM01978|I70160|1-99: V42-E130Potential Phosphorylation Sites: S38, S58, S117, S128, S170 MOTIFS 427526315CD1 386 Protein kinase domain: L16-V266 HMMER_PFAMSerine/Threonine protein kinases, catalytic domain: L16-L262 HMMER_SMARTProtein kinases signatures and profile: I107-T161 PROFILESCAN PROTEINKINASE DOMAIN BLAST_DOMO DM00004|A53800|119-368: E20-K221DM00004|A55318|159-389: D15-W216 DM00004|JC2363|126-356: D15-W216DM00004|Q05609|553-797: E20-S233 Potential Phosphorylation Sites: S61,S89, S96, S233, S273, S277, S295, S341, S346, S360, MOTIFS S365, T345,Y274 Potential Glycosylation Sites: N97, N159, N340 MOTIFS Leucinezipper pattern: L225-L246, L232-L253 MOTIFS Serine/Threonine proteinkinases active-site signature: V129-I141 MOTIFS 43 7526442CD1 152Eukaryotic protein kinase IPB000719: H119-Q134 BLIMPS_BLOCKS PROTEINKINASE DOMAIN BLAST_DOMO DM00004|I49592|6-276: L7-R131DM00004|P2343716-286: R9-R131 DM00004|P29620|21-289: I10-P130DM00004|Q02399|6-276: L7-R131 Protein kinases ATP-binding regionsignature: I10-K33 MOTIFS

TABLE 4 Poly- nucleotide SEQ ID NO:/ Incyte ID/ Sequence Length SequenceFragments 44/ 1-937, 1-1916, 479-1163, 762-1440, 765-1648, 1025-19167517831- CB1/ 1916 45/ 1-584, 1-755, 1-764, 2-925, 149-926, 218-9267520272- CB1/ 926 46/ 1-828, 530-1382, 591-1382, 635-1382, 989-13827521279- CB1/ 1382 47/ 1-665, 1-876, 2-1677, 158-1087, 167-1047,293-1057, 736-1536, 744-1267, 744-1526, 744-1593, 978-1678 7523965- CB1/1678 48/ 1-855, 2-812, 539-895 7524016- CB1/ 895 49/ 1-819, 2-764,510-1294 7524680- CB1/ 1294 50/ 1-647, 1-710, 2-677, 573-1354 7524757-CB1/ 1354 51/ 1-726, 1-1179, 300-1179, 349-1204, 396-1180, 474-1201,475-1201, 497-1201, 568-1201, 582-1201 7516229- CB1/ 1204 52/ 1-425,1-672, 1-701, 1-717, 1-935, 1-1859, 205-1118, 728-1623, 859-1858,888-1859, 974-1859, 1050-1858, 1053-1859, 1092-1851, 1114- 7516525-1859, 1130-1859, 1149-1859, 1167-1859, 1267-1858, 1289-1859 CB1/ 185953/ 1-767, 1-1692, 28-1025, 694-1645, 844-1695 7516533- CB1/ 1695 54/1-176, 1-805, 1-914, 1-3891, 231-3891, 613-1352, 642-1329, 643-1438,644-1447, 668-1289, 847-1618, 860-1623, 1380-2098, 1388-2109, 7516613-1541-1948, 1541-2000, 1558-1901, 1670-2559, 1872-2565, 1881-2589,2304-3236, 2433-3222, 2438-3215, 2442-3223, 2470-3204, 2549- CB1/ 38913214, 3007-3891, 3103-3891, 3270-3891, 3283-3891, 3337-3891 55/ 1-603,1-778, 1-3954, 410-1315, 416-1127, 440-798, 1037-2445, 1132-2069,1138-1925, 1623-2614, 1690-2567, 2379-3252, 2446-3954, 7517068-2613-3234, 3186-3954 CB1/ 3954 56/ 1-655, 26-3357, 377-1295, 447-1269,580-1302, 1190-1924, 1194-2055, 1230-1956, 1442-2298, 2241-2719,2545-3357, 2678-3356 7517148- CB1/ 3357 57/ 1-772, 1-2006, 418-1330,573-1330, 732-1615, 746-1617, 1195-2029, 1199-2036, 1207-2029,1268-2004, 1298-2036 7517238- CB1/ 2036 58/ 1-645, 26-2541, 529-1345,942-1833, 1752-2541, 1775-2541, 1950-2540 7518685- CB1/ 2541 59/ 1-802,1-803, 1-880, 1-2529, 208-1179, 464-1177, 707-1448, 732-1469, 1113-1952,1143-1948, 1692-2582, 1781-2555, 1858-2611, 1916- 7520192- 2555,2043-2555 CB1/ 2611 60/ 1-781, 1-830, 2-5215, 44-5215, 190-723,397-1135, 414-1120, 708-1383, 741-1569, 757-1546, 757-1606, 1186-2040,1191-2029, 1556- 7520428- 2392, 1561-2342, 1967-2655, 2006-2059,2120-2780, 2380-3164, 2407-3194, 2769-3419, 2771-3450, 3167-3823,3193-3831, 3562-4286, CB1/ 5216 3565-4101, 3633-4506, 3770-4506,3974-4724, 3975-4708, 4346-5082, 4352-4996, 4447-5215, 4448-5216,4450-5216, 4457-5216, 4460- 5216, 4478-5216, 4588-5216, 4771-5216,4795-5216, 5125-5216, 5130-5216 61/ 1-817, 1-2554, 655-1515, 678-1376,970-1849, 1110-1840, 1319-1729, 1462-2334, 1470-2177, 1470-2240,1490-2270, 1765-2554 7522586- CB1/ 2554 62/ 1-2022, 1035-1956,1163-1951, 1175-2022, 1178-2023 7524017- CB1/ 2023 63/ 1-847, 2-1128,184-1129 7525773- CB1/ 1129 64/ 1-687, 2-686 7525861- CB1/ 687 65/1-3912, 692-1142, 692-1191, 692-1374, 692-1383, 692-1385, 991-1569,1053-1112, 1118-1449, 1118-1619, 1118-1704, 1118-1717, 1118- 2509577-1774, 1193-1462, 1193-1505, 1193-1625, 1193-1667, 1193-1687, 1193-1702,1193-1716, 1193-1724, 1193-1735, 1202-1884, 1306-1624, CB1/ 39121308-1841, 1308-1866, 1308-1989, 1308-2001, 1308-2006, 1323-2016,1333-1833, 1831-2300, 1831-2302, 1838-2524, 1911-2622, 1921- 2796,2158-2951, 2231-2430, 2266-2685, 2269-2951, 2359-2951, 2441-2632,2441-2957, 2441-2965, 2466-3026, 2467-2943, 2475-3193, 2676-2925,2969-3225 66/ 1-2813, 147-2813, 1800-2249, 1976-2224, 1976-2590,2139-2331, 2290-2373, 2290-2484, 2290-2512, 2290-2573, 2290-2587,2290-2592, 7505222- 2290-2596, 2290-2601, 2290-2626, 2290-2636,2290-2641, 2290-2669, 2290-2673, 2290-2702, 2290-2732, 2290-2755,2290-2760, 2290- CB1/ 3229 2770, 2290-2776, 2290-2783, 2290-2825,2290-2861, 2290-2864, 2290-2871, 2290-2899, 2290-2981, 2295-2804,2315-2833, 2427-3094, 2427-3215, 2465-3204, 2482-3211, 2495-2899,2509-2900, 2536-3229, 2538-2900, 2575-2812, 2596-2819, 2649-3229 67/1-753, 1-877, 2-669, 2-676, 2-718, 2-2099, 529-1448, 548-1422, 631-1515,640-1476, 1257-2100, 1319-2100, 1340-2099, 1345-2100, 7524408- 1353-2099CB1/ 2100 68/ 1-718, 1-789, 11-4017, 14-590, 17-664, 17-674, 24-849,79-221, 169-4213, 171-849, 241-929, 311-946, 380-828, 421-1058,440-1069, 7526163- 466-1171, 494-984, 494-1004, 494-1017, 494-1019,494-1037, 494-1122, 494-1270, 494-1279, 509-1065, 541-1058, 559-1058,654- CB1/ 4213 1148, 674-1307, 676-1286, 722-874, 731-1267, 750-1460,850-1386, 940-1516, 960-1765, 963-1639, 965-1553, 965-1590, 973-1633,975-1571, 1023-1616, 1071-1647, 1079-1569, 1093-1716, 1101-1585,1110-1658, 1110-1689, 1110-1725, 1117-1590, 1120-1788, 1127- 1992,1134-1635, 1135-1581, 1144-1648, 1145-1876, 1157-1598, 1161-1839,1162-1772, 1196-1857, 1204-1762, 1207-1915, 1215-1877, 1262-1805,1277-1775, 1280-1981, 1290-1915, 1298-1711, 1303-1964, 1349-1825,1354-1973, 1367-2079, 1378-2014, 1384-2081, 1418-1946, 1422-1915,1427-1948, 1431-1980, 1433-2054, 1440-2126, 1460-2112, 1488-1898,1533-2187, 1544-2393, 1559-2390, 1570-2397, 1583-2386, 1608-2050,1612-2210, 1639-2204, 1639-2221, 1639-2291, 1639-2342, 1641-2322,1666-2393, 1676-2347, 1684-2366, 1692-2223, 1701-2393, 1704-2231,1706-2320, 1706-2393, 1711-2531, 1724-2431, 1755-2310, 1760-2393,1762-2192, 1763-2192, 1771-2398, 1793- 2259, 1798-2393, 1809-2393,1813-2393, 1816-2393, 1818-2333, 1820-2393, 1856-2393, 1858-2393,1885-2393, 1897-2393, 1913-2393, 1915-2393, 1916-2393, 1936-2393,1937-2393, 1980-2644, 1993-2393, 2005-2393, 2021-2484, 2029-2419,2086-2393, 2148-2792, 2156- 2180, 2276-3024, 2289-3020, 2314-3220,2329-2786, 2329-2829, 2351-2940, 2363-3123, 2586-3050, 2602-3014,2628-3274, 2696-3341, 2731-3244, 2840-3377, 2863-3679, 2945-3400,3402-3605, 3428-3935, 3595-4034, 3627-4183, 3632-4061, 3638-4091,3711-4185, 3720- 4189, 3809-4191, 3809-4212, 3909-4160, 3909-4181,4059-4213 69/ 1-882, 1-5991, 20-942, 36-335, 66-942, 99-314, 133-335,175-942, 214-942, 241-334, 262-4306, 333-941, 333-942, 4041039, 473-921,7526158- 473-932, 514-1151, 533-1162, 559-1264, 587-1077, 587-1097,587-1110, 587-1112, 587-1130, 587-1215, 587-1363, 587-1372, 602-1158,CB1/ 5991 634-1151, 652-1151, 747-1241, 1068-1664, 1172-1662, 1186-1809,1194-1678, 1213-1881, 1220-2085, 1238-1969, 1254-1932, 1297- 1855,1370-1868, 1373-2074, 1391-1804, 1442-1918, 1471-2107, 1477-2174,1511-2039, 1515-2008, 1520-2041, 1524-2073, 1533-2219, 1553-2205,1581-1991, 1626-2280, 1637-2486, 1652-2483, 1663-2490, 1676-2479,1701-2143, 1705-2303, 1732-2297, 1732-2314, 1732-2384, 1732-2435,1734-2415, 1759-2486, 1769-2440, 1777-2459, 1785-2316, 1794-2486,1797-2324, 1799-2413, 1799-2486, 1804- 2624, 1817-2524, 1848-2403,1853-2486, 1864-2491, 1886-2352, 1891-2486, 1902-2486, 1906-2486,1909-2486, 1913-2486, 1949-2486, 1951-2486, 1978-2486, 1990-2486,2006-2486, 2008-2486, 2009-2486, 2029-2486, 2030-2486, 2073-2737,2086-2486, 2098-2486, 2114-2577, 2122-2512, 2179-2486, 2241-2885,2369-3117, 2382-3113, 2407-3313, 2422-2879, 2422-2922, 2444-3033,2456-3216, 2679-3143, 2695- 3107, 2721-3367, 2789-3434, 2824-3337,2933-3470, 2956-3772, 3038-3493, 3495-3698, 3521-4028, 3720-4276,3725-4154, 3731-4184, 3740-4127, 3804-4278, 3813-4282, 3902-4289,3902-4305, 4002-4253, 4002-4274, 4152-4306, 4403-4463, 4537-4598 70/1-669 7519807- CB1/ 669 71/ 1-224, 1-361, 1-376, 1-409, 1-416, 1-432,1-437, 1-443, 1-445, 1-455, 1-457, 1-483, 1-2049, 2-193, 2-424, 2-464,2-504, 3-358, 3-419, 3- 7526180- 518, 4-190, 4-280, 7-860, 10-561,10-579, 10-637, 14-445, 17-509, 34-331, 37-732, 37-762, 37-782, 37-784,37-896, 39-695, 39-726, 39- CB1/ 2453 758, 39-782, 39-814, 39-880,40-808, 42-572, 53-804, 71-320, 71-699, 84-293, 85-614, 107-526,120-572, 131-748, 141-420, 151-509, 154-525, 155-806, 182-475, 182-666,186-525, 188-512, 188-525, 188-733, 203-504, 219-504, 219-525, 233-518,233-630, 242-331, 248-699, 259-444, 260-614, 260-621, 260-653, 260-748,260-749, 261-627, 273-840, 277-614, 353-568, 358-1005, 398-575,401-1853, 426-650, 426-656, 426-800, 426-911, 426-1090, 432-1071,434-690, 435-598, 439-1223, 444-693, 444-717, 444-767, 449-718, 451-637,452-1071, 454-643, 454-1281, 454-1458, 459-1283, 463-981, 467-1018,468-1168, 486-715, 493-699, 499-1095, 508-764, 508-1050, 511-1137,527-778, 527-1031, 527-1032, 533-1049, 533-1171, 534-1033, 534-1049,549-1095, 550-929, 575-1095, 580-1027, 593-1072, 595-767, 605-1031,618-890, 618-1131, 620-798, 638-1335, 639-1032, 650-1105, 650-1133,667-1200, 667-1207, 667- 1227, 667-1251, 672-1176, 673-727, 678-1135,679-975, 684-968, 711-1111, 718-1530, 724-1396, 724-1532, 724-1547,726-1437, 738- 1115, 739-1108, 739-1110, 747-1054, 749-1051, 749-1161,752-1553, 774-1015, 774-1054, 790-1207, 792-1048, 792-1054, 792-1109,792-1131, 792-1161, 792-1228, 792-1298, 793-1132, 801-1468, 817-1554,838-1477, 842-1207, 843-1144, 849-1118, 854-1530, 865- 1095, 885-1414,889-1393, 903-1041, 903-1406, 905-1548, 907-1081, 920-1136, 932-1613,939-1111, 939-1263, 945-1391, 950-1273, 973-1328, 1050-1429, 1060-1635,1071-1147, 1076-1376, 1099-1558, 1120-1512, 1127-1259, 1147-1573,1179-1878, 1194-1716, 1215- 1622, 1222-1622, 1223-1478, 1241-1558,1250-1739, 1259-2177, 1266-1671, 1271-1524, 1276-1544, 1284-1622,1295-1554, 1299-1561, 1299-1573, 1325-1946, 1381-1619, 1381-1642,1398-1601, 1398-1622, 1438-2095, 1443-1743, 1444-1558, 1453-1656,1473-1704, 1484-1753, 1523-1573, 1531-1917, 1578-1763, 1578-1904,1578-2059, 1578-2062, 1578-2063, 1578-2064, 1581-1999, 1581-2112,1589-2045, 1594-1931, 1606-2042, 1616-2049, 1631-1867, 1672-2453,1685-2408, 1697-2048, 1719-1980, 1742-2000, 1744-2112, 1784- 2095,1788-2408, 1796-2054, 1805-2095, 1817-2372, 1855-2408, 1869-2167,1880-2071; 1880-2087, 1880-2091, 1880-2092, 1880-2097, 1880-2315,1883-2353, 1905-2117, 1926-2141, 1941-2110, 1946-2054, 1951-2169,1971-2408, 1971-2421, 1979-2408, 1980-2408, 1994- 2408 72/ 1-4430,313-1222, 358-592, 649-4430, 938-1574, 1084-1754, 1095-1765, 1185-1978,1198-1884, 1204-1880, 1208-1859, 1214-2029, 7526185- 1223-2017,1255-1917, 1255-2004, 1259-1957, 1282-2255, 1298-1950, 1321-2017,1335-2004, 1352-2002, 1354-2004, 1383-2002, 1396- CB1/ 4430 2064,1469-2171, 1503-2297, 1506-2183, 1515-2243, 1528-2457, 1546-2439,1560-2428, 1560-2458, 1582-2282, 1582-2429, 1587-2305, 1599-2338,1605-2350, 1625-2433, 1638-2413, 1640-2392, 1666-2445, 1693-2395,1716-2469, 1718-2467, 1720-2469, 1724-2451, 1724- 2463, 1724-2467,1772-2468, 1775-2468, 1782-2468, 1782-2469, 1783-2431, 1786-2468,1801-2467, 1809-2468, 1816-2469, 1829-2469, 1833-2469, 1852-2469,1858-2469, 2196-2847, 2386-3249, 3402-4093, 3471-4274, 3698-4426 73/1-3276, 734-1407, 1223-1711, 1860-2512, 1860-2524, 1925-2468, 2447-3057,2552-3009 7526192- CB1/ 3276 74/ 1-344, 20-367, 20-3910, 99-612,340-775, 464-775, 486-797, 500-746, 520-779, 537-799, 552-768, 560-779,567-790, 582-1094, 587- 7526193- 1213, 610-895, 616-1173, 634-1307,698-1094, 706-1162, 776-1097, 778-1097, 797-1101, 799-1093,805-1348,..824-1456, 824-1504, CB1/ 3910 825-1466, 830-1503, 834-1412,840-1124, 840-1426, 849-1097, 868-1096, 885-1348, 1048-1744, 1115-1714,1124-1734, 1127-1742, 1146-1711, 1217-1481, 1222-1524, 1246-1525,1249-1757, 1251-1509, 1295-1751, 1321-1832, 1334-1889, 1376-1647,1376-1670, 1376-1768, 1387-1768, 1466-1742, 1470-1829, 1491-2092,1497-1880, 1502-1820, 1520-2235, 1599-2231, 1961-1989, 1992-2298,2014-2654, 2076-2336, 2092-2727, 2135-2425, 2135-2448, 2147-2580,2153-2443, 2226-2705, 2476-2624, 2508-2889, 2511-2969, 2530-2930,2807-3117, 2862-3393, 2870-3085, 3117-3543, 3343-3863, 3458-3789,3569-3807 75/ 1-4380, 41-683, 763-1641, 818-1676, 884-1835, 887-1743,887-1834, 887-1835, 897-1740, 900-1709, 954-1835, 1032-1590, 1035-1895,7526196- 1036-1729, 1036-1835, 1039-1835, 1065-1676, 1141-1738,1169-1738, 1176-1738, 1188-1738, 1189-1737, 1198-1920, 1198-2052, 1202-CB1/ 4380 1738, 1304-1944, 1353-2006, 1354-1879, 1354-1985, 1354-2016,1354-2051, 1354-2086, 1356-2011, 1411-2084, 1411-2094, 1498-2168,2279-2898, 2279-2901, 2279-2918, 2279-2945, 2279-2987, 2279-3041,2279-3091, 2279-3111, 2279-3151, 2285-3136, 2361-2898, 2365- 3037,2368-3037, 2392-2942, 2398-3036, 2406-2999, 2422-3076, 2429-3046,2430-3037, 2447-3014, 2500-3037, 2538-3118, 3013-3077, 3108-4157 76/1-622, 1-624, 1-4163, 3-677, 49-748, 76-607, 83-981, 86-731, 86-826,101-650, 102-660, 102-693, 138-660, 138-847, 139-730, 139-807, 7526198-139-833, 139-847, 139-848, 139-914, 139-926, 139-1002, 194827, 299-882,318-882, 2082-2933, 2092-2695, 2092-2698, 2092-2715, CB1/ 42932092-2742, 2092-2784, 2092-2838, 2092-2888, 2092-2908, 2092-2948,2158-2695, 2162-2834, 2165-2834, 2189-2739, 2195-2833, 2203-2796,2219-2873, 2226-2843, 2227-2834, 2244-2811, 2297-2834, 2335-2915,2810-2874, 2905-3964, 3908-4293 77/ 1-581, 21-6538, 438-1053, 581-1057,589-1057, 906-1436, 1332-1978, 1483-2045, 1486-2135, 1645-2142,2127-2864, 2129-2864, 2304- 7526208- 2751, 2382-2971, 2543-3089,2564-3118, 2568-3068, 3104-3725, 3255-3883, 3376-3897, 3450-4136,3459-4057, 3476-3968, 3478-4027, CB1/ 6538 3479-4046, 3481-4172,3489-4296, 3502-4173, 3522-4172, 3533-4165, 3555-4177, 3564-4310,3578-4049, 3643-4157, 3643-4275, 3652- 4172, 3663-4244, 3676-4334,3699-4147, 3806-4475, 3959-4517, 3972-4672, 4006-4691, 4007-4745,4028-4745, 4040-4746, 4043-4695, 4067-4745, 4076-4745, 4078-4745,4104-4659, 4116-4745, 4155-4745, 4165-4745, 4184-4745, 4194-4686,4195-4685, 4215-4689, 4216- 4688, 4242-4745, 4251-4702, 4284-4745,4873-5519, 4878-5586, 5089-5766, 5144-5808, 5483-6067, 5491-6053,5502-6329, 5571-6067, 5580-6040, 5586-6065, 5587-6067, 5587-6069,5603-6069, 5614-6067 78/ 1-581, 1-2290, 438-1053, 581-1057, 589-1057,906-1436, 1332-1978, 1483-2045, 1608-2349 7526212- CB1/ 2349 79/ 1-8009,203-877, 203-936, 571-1314, 571-1436, 571-1450, 577-1378, 586-1449,594-1332, 652-1450, 731-1652, 747-1525, 756-1532, 7526213- 799-1670,806-1686, 833-1552, 833-1695, 833-1721, 833-1768, 843-1675, 849-1612,850-1644, 850-1675, 855-1615, 856-1771, 857- CB1/ 8015 1624, 857-1678,915-1764, 928-1675, 928-1768, 928-1899, 933-1753, 933-1779, 934-1774,943-1759, 947-1898, 950-1844, 1827-2719, 1829-2642, 1829-2704,1829-2737, 1830-2720, 2108-3060, 2154-3036, 2155-3043, 2160-3084,2161-3052, 2172-3054, 2174-3015, 2180- 2972, 2212-3066, 2212-3163,2215-2958, 2215-3030, 2277-3051, 2281-3068, 2422-3338, 2430-3130,2430-3212, 2430-3244, 2430-3322, 2434-3184, 5975-6837, 6557-7314,7215-7903, 7241-8015 80/ 1-7945, 203-668, 203-955, 492-1352, 507-1304,507-1371, 507-1385, 513-1313, 514-1304, 522-1384, 588-1385, 692-1396,707-787, 735- 7526214- 1548, 742-1621, 769-1487, 769-1656, 769-1703,785-1551, 786-1610, 787-1550, 787-1559, 788-1706, 790-1712, 791-1488,792-1613, CB1/ 7945 1796-676, 863-1610, 863-1703, 863-1834, 868-1688,868-1714, 869-1709, 878-1694, 882-1833, 885-1779, 1762-2657, 1764-2579,1764- 2642, 1764-2675, 1765-2658, 2418-3318, 2583-3356, 2664-3334,2664-3380, 2664-3382, 2664-3389, 2664-3405, 2664-3422, 2664-3431,2664-3445, 2664-3454, 2664-3458, 2664-3459, 2664-3464, 2664-3469,2664-3470, 2664-3471, 2664-3480, 2664-3510, 2664-3511, 2664- 3532,2664-3562, 2668-3458, 2668-3609, 2668-3617, 2706-3617, 2711-3617,2776-3617, 2779-3617, 2781-3654, 2782-3617, 2784-3654, 2804-3617,2810-3654, 2823-3653, 2848-3654, 2849-3628, 2849-3651, 2849-3654,28543654, 2876-3653, 2876-3654, 2899-3650, 2906- 3654, 2915-3654,2949-3654, 2952-3654, 2971-3654, 3008-3682, 3181-3922, 3471-4320,3797-4551, 4066-4870, 4320-5134, 4541-5355, 4764-5490, 5247-5934,5910-6772, 6492-7250, 6854-7555, 7150-7839, 7176-7945 81/ 1-528, 1-719,30-3149, 217-706, 229-740, 239-1013, 243-910, 263-996, 402-1249,1515-2354, 1538-2050, 1569-2354, 1889-2602, 2023- 7526228- 2773,2112-2648, 2124-2819, 2147-2671, 2211-2688, 2566-3119 CB1/ 3149 82/1-563, 118-3617, 407-1087, 412-1145, 506-1090, 521-563, 703-1270,869-1158, 869-1501, 1194-1870, 1194-1985, 1194-1992, 1358- 7526246-2314, 1371-2039, 1373-2314, 1376-2303, 1385-2314, 1421-2000, 1423-2016,1423-2314, 1429-2101, 1439-2254, 1455-2314, 1457-2209, CB1/ 36171466-2314, 1471-2313, 1471-2314, 1478-2314, 1480-2314, 1492-2313,1494-2313, 1494-2314, 1495-2351, 1497-2314, 1497-2351, 1509- 2150,1509-2351, 1515-2314, 1519-2303, 1520-2314, 1521-2249, 1529-2314,1539-2152, 1542-2130, 1542-2336, 1542-2367, 1542-2412, 1544-2314,1545-2247, 1557-2187, 1568-2313, 1568-2314, 1592-2177, 1594-2303,1598-2314, 1599-2282, 1599-2351, 1601-2189, 1613- 2314, 1616-2267,1616-2314, 1625-2252, 1631-2190, 1638-2313, 1648-2404, 1653-2261,1656-2242, 1661-2344, 1677-2397, 1679-2352, 1688-2368, 1714-2320,1715-2528, 1728-2280, 1737-2340, 1740-2392, 1744-2314, 1773-2465,1782-2351, 1797-2414, 1824-2428, 2599- 3242 83/ 1-1955, 81-946,715-1172, 812-1528, 987-1540 7526258- CB1/ 1955 84/ 1-2937, 122-658,122-690, 122-739, 123-1393, 539-1393, 621-1393, 807-1393, 1099-1644,1237-1848, 1251-1561, 1390-2042, 1392- 7526311- 2069, 1396-2052,1430-1987, 1495-1949, 1502-1940, 1531-2266, 1542-2039, 1549-2106,1579-1965, 1658-2132, 1731-2380, 1737-2490, CB1/ 2937 1759-2540,1837-2534, 1853-2606, 1871-2301, 1880-2223, 1925-2496, 1960-2513,1971-2483, 1984-2421, 1984-2491, 1984-2567, 1984- 2611, 1992-2496,2000-2404, 2015-2565, 2021-2502, 2033-2567, 2038-2544, 2060-2394,2062-2561, 2066-2606, 2083-2510, 2107-2572, 2109-2500, 2129-2573,2136-2565, 2139-2582, 2147-2567, 2154-2547, 2156-2606, 2160-2609,2169-2741, 2182-2609, 2223-2552, 2240-2586, 2246-2606, 2247-2568,2247-2602, 2328-2878, 2355-2860, 2374-2890, 2472-2913, 2478-2937 85/1-6121, 193-871, 310-931, 573-982, 867-1481, 867-1492, 867-1496,867-1573, 867-1595, 890-1609, 919-1643, 922-1655, 1103-1948, 7526315-2143-2950, 2339-3054, 2423-3023, 2471-3073, 2669-3237, 2738-3490,2738-3554, 2800-3574, 3049-3795, 3247-3955, 4679-5357, 4789- CB1/ 61225413, 5055-5683, 5121-5809, 5167-5854, 5317-6012, 5343-6045, 5356-6064,5370-6043, 5370-6116, 5405-6106, 5410-6122, 5424-6117, 5458-6108,5461-6122, 5479-6079, 5493-6117, 5495-6063, 5524-6117 86/ 1-437, 1-647,1-1893, 1-1914, 260-862, 260-863, 684-1206, 684-1379, 720-1262,741-1452, 744-1453, 748-1386, 750-1276, 760-1283, 7526442- 762-1262,784-1394, 789-1234, 789-1378, 791-1267, 794-1261, 796-1623, 807-1405,814-1325, 818-1406, 821-1623, 845-1278, 845- CB1/ 1914 1332, 856-1377,863-1451, 876-1423, 880-1464, 953-1403, 960-1772, 961-1395, 969-1452,1474-1914

TABLE 5 Polynucleotide SEQ ID NO: Incyte Project ID: RepresentativeLibrary 65 2509577CB1 TESTNOC01 66 7505222CB1 LUNGDIN02 68 7526163CB1BRAITDR02 69 7526158CB1 THPITXT04 71 7526180CB1 BRSTNOT01 72 7526185CB1UTREDMF02 73 7526192CB1 NERDTDN03 74 7526193CB1 BRABDIR01 75 7526196CB1BRACNOK02 76 7526198CB1 BRACNOK02 77 7526208CB1 BLADDIT01 78 7526212CB1BRAINOR03 80 7526214CB1 MYEPUNN01 81 7526228CB1 MYEPUNF01 82 7526246CB1THYMNOE01 83 7526258CB1 BLYRTXT03 84 7526311CB1 SININOT04 85 7526315CB1OVARDIN02 86 7526442CB1 PITUNON01

TABLE 6 Library Vector Library Description BLADDIT01 pINCY Library wasconstructed using RNA isolated from diseased bladder tissue removed froma 73-year-old male during a total cystectomy. Pathology indicated thebladder mucosa showed mild chronic cystitis. Pathology for theassociated tumor tissue indicated invasive grade 3 adenocarcinoma, whichformed a friable mass situated within the proximal urethra, 14 cm fromthe distal urethral resection margin. The tumor invaded superficiallyinto, but not through, muscularis propria. BLYRTXT03 pINCY Library wasconstructed using RNA isolated from a treated Raji cell line derivedfrom the B-lymphocyte cells of an 11-year-old Black male (ATCC CCL-86).The cells were treated for 18 hours with 10 ng/ml of interleukin 18(IL-18). Pathology indicated Burkitt's lymphoma. BRABDIR01 pINCY Librarywas constructed using RNA isolated from diseased cerebellum tissueremoved from the brain of a 57-year- old Caucasian male, who died from acerebrovascular accident. Patient history included Huntington's disease,emphysema, and tobacco abuse. BRACNOK02 PSPORT1 This amplified andnormalized library was constructed using RNA isolated from posteriorcingulate tissue removed from an 85-year-old Caucasian female who diedfrom myocardial infarction and retroperitoneal hemorrhage. Pathologyindicated atherosclerosis, moderate to severe, involving the circle ofWillis, middle cerebral, basilar and vertebral arteries; infarction,remote, left dentate nucleus; and amyloid plaque deposition consistentwith age. There was mild to moderate leptomeningeal fibrosis, especiallyover the convexity of the frontal lobe. There was mild generalizedatrophy involving all lobes. The white matter was mildly thinned.Cortical thickness in the temporal lobes, both maximal and minimal, wasslightly reduced. The substantia nigra pars compacta appeared mildlydepigmented. Patient history included COPD, hypertension, and recurrentdeep venous thrombosis. 6.4 million independent clones from thisamplified library were normalized in one round using conditions adaptedfrom Soares et al., PNAS (1994) 91:9228-9232 and Bonaldo et al., GenomeResearch 6 (1996):791. BRAINOR03 PBK-CMV This random primed library wasconstructed using pooled cDNA from two donors. cDNA was generated usingmRNA isolated from brain tissue removed from a Caucasian male fetus(donor A) who was stillborn with a hypoplastic left heart at 23 weeks'gestation and from brain tissue removed from a Caucasian male fetus(donor B), who died at 23 weeks' gestation from premature birth.Serologies were negative for both donors and family history for donor Bincluded diabetes in the mother. BRAITDR02 PCDNA2.1 This random primedlibrary was constructed using RNA isolated from allocortex, neocortex,anterior and frontal cingulate tissue removed from a 55-year-oldCaucasian female who died from cholangiocarcinoma. Pathology indicatedmild meningeal fibrosis predominately over the convexities, scatteredaxonal spheroids in the white matter of the cingulate cortex and thethalamus, and a few scattered neurofibrillary tangles in the entorhinalcortex and the periaqueductal gray region. Pathology for the associatedtumor tissue indicated well-differentiated cholangiocarcinoma of theliver with residual or relapsed tumor. Patient history includedcholangiocarcinoma, post-operative Budd-Chiari syndrome, biliaryascites, hydrothorax, dehydration, malnutrition, oliguria and acuterenal failure. Previous surgeries included cholecystectomy and resectionof 85% of the liver. BRSTNOT01 PBLUESCRIPT Library was constructed usingRNA isolated from the breast tissue of a 56-year-old Caucasian femalewho died in a motor vehicle accident. LUNGDIN02 pINCY This normalizedlung tissue library was constructed from 7.6 million independent clonesfrom a diseased lung tissue library. Starting RNA was made from RNAisolated from diseased lung tissue. Pathology indicated ideopathicpulmonary disease. The library was normalized in 2 rounds usingconditions adapted from Soares et al., PNAS (1994) 91:9228-9232 andBonaldo et al., Genome Research 6 (1996):791, except that asignificantly longer (48 hours/round) reannealing hybridization wasused. MYEPUNF01 pRARE This 5′ cap isolated full-length library wasconstructed using RNA isolated from an untreated K-562 cell line,derived from chronic myelogenous leukemia precursor cells removed from a53-year-old female. MYEPUNN01 pRARE This normalized untreated K-562 cellline tissue library was constructed from independent clones from a K-562cell line library. Starting RNA was made from an untreated K-562 cellline, derived from chronic myelogenous leukemia precursor cells removedfrom a 53-year-old female. The library was normalized in one round usingconditions adapted from Soares et al., PNAS (1994) 91:9228-9232 andBonaldo et al., Genome Research 6 (1996):791, except that asignificantly longer (48 hours/round) reannealing hybridization wasused. NERDTDN03 pINCY This normalized dorsal root ganglion tissuelibrary was constructed from 1.05 million independent clones from adorsal root ganglion tissue library. Starting RNA was made from dorsalroot ganglion tissue removed from the cervical spine of a 32-year-oldCaucasian male who died from acute pulmonary edema, acutebronchopneumonia, bilateral pleural effusions, pericardial effusion, andmalignant lymphoma (natural killer cell type). The patient presentedwith pyrexia of unknown origin, malaise, fatigue, and gastrointestinalbleeding. Patient history included probable cytomegalovirus infection,liver congestion, and steatosis, splenomegaly, hemorrhagic cystitis,thyroid hemorrhage, respiratory failure, pneumonia of the left lung,natural killer cell lymphoma of the pharynx, Bell's palsy, and tobaccoand alcohol abuse. Previous surgeries included colonoscopy, closed colonbiopsy, adenotonsillectomy, and nasopharyngeal endoscopy and biopsy.Patient medications included Diflucan (fluconazole), Deltasone(prednisone), hydrocodone, Lortab, Alprazolam, Reazodone,ProMace-Cytabom, Etoposide, Cisplatin, Cytarabine, and dexamethasone.The patient received radiation therapy and multiple blood transfusions.The library was normalized in 2 rounds using conditions adapted fromSoares et al., PNAS (1994) 91:9228-9232 and Bonaldo et al., GenomeResearch 6 (1996):791, except that a significantly longer (48 hours/round) reannealing hybridization was used. OVARDIN02 pINCY Thisnormalized ovarian tissue library was constructed from 5.76 millionindependent clones from an ovary library. Starting RNA was made fromdiseased ovarian tissue removed from a 39-year-old Caucasian femaleduring total abdominal hysterectomy, bilateral salpingo-oophorectomy,dilation and curettage, partial colectomy, incidental appendectomy, andtemporary colostomy. Pathology indicated the right and left adnexa,mesentery and muscularis propria of the sigmoid colon were extensivelyinvolved by endometriosis. Endometriosis also involved the anterior andposterior serosal surfaces of the uterus and the cul-de-sac. Theendometrium was proliferative. Pathology for the associated tumor tissueindicated multiple (3 intramural, 1 subserosal) leiomyomata. The patientpresented with abdominal pain and infertility. Patient history includedscoliosis. Family history included hyperlipidemia, benign hypertension,atherosclerotic coronary artery disease, depressive disorder, braincancer, and type II diabetes. The library was normalized in two roundsusing conditions adapted from Soares et al., PNAS(1994) 91:9228 andBonaldo et al., Genome Research 6 (1996):791, except that asignificantly longer (48-hours/round) reannealing hybridization wasused. PITUNON01 pINCY This normalized pituitary gland tissue library wasconstructed from 6.92 million independent clones from a pituitary glandtissue library. Starting RNA was made from pituitary gland tissueremoved from a 55-year-old male who died from chronic obstructivepulmonary disease. Neuropathology indicated there were no grossabnormalities, other than mild ventricular enlargement. There was noapparent microscopic abnormality in any of the neocortical areasexamined, except for a number of silver positive neurons with apicaldendrite staining, particularly in the frontal lobe. The significance ofthis was undetermined. The only other microscopic abnormality was thatthere was prominent silver staining with some swollen axons in the CA3region of the anterior and posterior hippocampus. Microscopic sectionsof the cerebellum revealed mild Bergmann's gliosis in the Purkinje celllayer. Patient history included schizophrenia. The library wasnormalized in two rounds using conditions adapted from Soares et al.,PNAS (1994) 91:9228-9232 and Bonaldo et al., Genome Research (1996)6:791, except that a significantly longer (48 hours/round) reannealinghybridization was used. SININOT04 pINCY Library was constructed usingRNA isolated from diseased ileum tissue obtained from a 26-year-oldCaucasian male during a partial colectomy, permanent colostomy, and anincidental appendectomy. Pathology indicated moderately to severelyactive Crohn's disease. Family history included enteritis of the smallintestine. TESTNOC01 PBLUESCRIPT This large size fractionated librarywas constructed using RNA isolated from testicular tissue removed from apool of eleven, 10 to 61-year-old Caucasian males. THP1TXT04 pINCYLibrary was constructed using RNA isolated from stimulated THP-1 cells.THP-1 is a human promonocyte line derived from the peripheral blood of a1-year-old male (Abbott Sample) with acute monocytic leukemia (Int. J.Cancer 26 (1980):171). THYMNOE01 PCDNA2.1 This 5′ biased random primedlibrary was constructed using RNA isolated from thymus tissue removedfrom a 2- year-old Caucasian female during a thymectomy and patchclosure of left atrioventricular fistula. Pathology indicated there wasno gross abnormality of the thymus. The patient presented withcongenital heart abnormalities. Patient history included double inletleft ventricle and a rudimentary right ventricle, pulmonaryhypertension, cyanosis, subaortic stenosis, seizures, and a fracture ofthe skull base. Patient medications included Lasix and Captopril. Familyhistory included reflux neuropathy in the mother. UTREDMF02 PCMV-ICISThis full-length enriched library was constructed using 1.5 microgramsof polyA RNA isolated from endometrial tissue removed from a 32-year-oldfemale. The endometrium was in secretory phase.

TABLE 7 Program Description Reference Parameter Threshold ABI A programthat removes vector sequences and masks Applied Biosystems, Foster City,CA. FACTURA ambiguous bases in nucleic acid sequences. ABI/ A Fast DataFinder useful in comparing and annotating Applied Biosystems, FosterCity, CA; Paracel Mismatch <50% PARACEL amino acid or nucleic acidsequences. Inc., Pasadena, CA. FDF ABI A program that assembles nucleicacid sequences. Applied Biosystems, Foster City, CA. AutoAssembler BLASTA Basic Local Alignment Search Tool useful in Altschul, S. F. et al.(1990) J. Mol. Biol. ESTs: Probability value = sequence similaritysearch for amino acid and nucleic 215:403-410; Altschul, S. F. et al.(1997) 1.0E−8 or less; Full Length acid sequences. BLAST includes fivefunctions: Nucleic Acids Res. 25:3389-3402. sequences: Probability value= blastp, blastn, blastx, tblastn, and tblastx. 1.0E−10 or less FASTA APearson and Lipman algorithm that searches for Pearson, W. R. and D. J.Lipman (1988) Proc. ESTs: fasta E value = similarity between a querysequence and a group of Natl. Acad Sci. USA 85:2444-2448; Pearson,1.06E−6; Assembled ESTs: sequences of the same type. FASTA comprises asW. R. (1990) Methods Enzymol. 183:63-98; fasta Identity = 95% or leastfive functions: fasta, tfasta, fastx, tfastx, and and Smith, T. F. andM. S. Waterman (1981) greater and Match length = ssearch. Adv. Appl.Math. 2:482-489. 200 bases or greater; fastx E value = 1.0E−8 or less;Full Length sequences: fastx score = 100 or BLIMPS A BLocks IMProvedSearcher that matches a sequence Henikoff, S. and J. G. Henikoff (1991)Nucleic Probability value = 1.0E−3 against those in BLOCKS, PRINTS,DOMO, Acids Res. 19:6565-6572; Henikoff, J. G. and or less PRODOM, andPFAM databases to search for gene S. Henikoff (1996) Methods Enzymol.266:88- families, sequence homology, and structural fingerprint 105; andAttwood, T. K. et al. (1997) J. Chem, regions. Inf. Comput. Sci.37:417-424. HMMER An algorithm for searching a query sequence againstKrogh, A. et al. (1994) J. Mol. Biol. 235:1501- PFAM, INCY, SMART orhidden Markov model (HMM)-based databases of 1531; Sonnhammer, E. L. L.et al. (1988) TIGRFAM hits: Probability protein family consensussequences, such as PFAM, Nucleic Acids Res. 26:320-322; Durbin, R. etvalue = 1.0E−3 or less; Signal INCY, SMART and TIGRFAM. al. (1998) OurWorld View, in a Nutshell, peptide hits: Score = 0 or Cambridge Univ.Press, pp. 1-350. greater ProfileScan An algorithm that searches forstructural and sequence Gribskov, M. et al. (1988) CABIOS 4:61-66;Normalized quality score ≧ motifs in protein sequences that matchsequence Gribskov, M. et al. (1989) Methods Enzymol. GCG-specified“HIGH” value patterns defined in Prosite. 183:146-159; Bairoch, A. etal. (1997) Nucleic for that particular Prosite Acids Res. 25:217-221.motif. Generally, score = 1.4-2.1. Phred A base-calling algorithm thatexamines automated Ewing, B. et al. (1998) Genome Res. 8:175- sequencertraces with high sensitivity and probability. 185; Ewing, B. and P.Green (1998) Genome Res. 8:186-194. Phrap A Phils Revised AssemblyProgram including SWAT Smith, T. F. and M. S. Waterman (1981) Adv. Score= 120 or greater; Match and CrossMatch, programs based on efficientAppl. Math. 2:482-489; Smith, T. F. and M. S. length = 56 or greaterimplementation of the Smith-Waterman algorithm, Waterman (1981) J. Mol.Biol. 147:195-197; useful in searching sequence homology and assemblingand Green, P., University of Washington, DNA sequences. Seattle, WA.Consed A graphical tool for viewing and editing Phrap Gordon, D. et al.(1998) Genome Res. 8:195- assemblies. 202. SPScan A weight matrixanalysis program that scans protein Nielson, H. et al. (1997) ProteinEngineering Score = 3.5 or greater sequences for the presence ofsecretory signal 10:1-6; Claverie, J. M. and S. Audic (1997) peptides.CABIOS 12:431-439. TMAP A program that uses weight matrices to delineatePersson, B. and P. Argos (1994) J. Mol. Biol. transmembrane segments onprotein sequences and 237:182-192; Persson, B. and P. Argos (1996)determine orientation. Protein Sci. 5:363-371. TMHMMER A program thatuses a hidden Markov model (HMM) Sonnhammer, E. L. et al. (1998) Proc.Sixth to delineate transmembrane segments on protein Intl. Conf. OnIntelligent Systems for Mol. sequences and determine orientation. Biol.,Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence (AAAI)Press, Menlo Park, CA, and MIT Press, Cambridge, MA, pp. 175-182. MotifsA program that searches amino acid sequences for Bairoch, A. et al.(1997) Nucleic Acids Res. patterns that matched those defined inProsite. 25:217-221; Wisconsin Package Program Manual, version 9, pageM51-59, Genetics Computer Group, Madison, WI.

TABLE 8 SEQ Caucasian African Asian Hispanic ID EST CB1 EST AlleleAllele Allele 1 Allele 1 Allele 1 Allele 1 NO: PID EST ID SNP ID SNP SNPAllele 1 2 Amino Acid frequency frequency frequency frequency 44 7517831142314T6 SNP00003755 149 1721 A A G noncoding n/a n/a n/a n/a 44 7517831142314T6 SNP00098537 3 1867 C C T noncoding n/a n/a n/a n/a 44 7517831142314T6 SNP00149767 132 1738 A A G noncoding n/a n/a n/a n/a 44 75178311531602H1 SNP00023921 178 1271 T T G noncoding n/d n/a n/a n/a 447517831 2655558T6 SNP00003755 126 1744 G A G noncoding n/a n/a n/a n/a44 7517831 2655558T6 SNP00149767 109 1761 A A G noncoding n/a n/a n/an/a 44 7517831 2829606H1 SNP00027387 110 109 G G A D18 n/a n/a n/a n/a44 7517831 2836842T6 SNP00003755 140 1730 A A G noncoding n/a n/a n/an/a 44 7517831 2836842T6 SNP00149767 123 1747 A A G noncoding n/a n/an/a n/a 44 7517831 2876073T6 SNP00003755 115 1755 A A G noncoding n/an/a n/a. n/a 44 7517831 2876073T6 SNP00149767 98 1772 A A G noncodingn/a n/a n/a n/a 44 7517831 7758626H1 SNP00126822 384 458 C C A noncodingn/a n/a n/a n/a 45 7520272 1265056T6 SNP00065601 250 838 A A G G274 n/an/a n/a n/a 45 7520272 1501560T6 SNP00069832 52 916 C C T noncoding n/an/a n/a n/a 45 7520272 1501560T6 SNP00075533 128 840 C C T S275 n/d n/an/a n/a 45 7520272 1968576T6 SNP00075533 204 866 C C T Q284 n/d n/a n/an/a 47 7523965 1238421H1 SNP00075756 56 794 G G A noncoding n/a n/a n/an/a 47 7523965 1324236T6 SNP00033242 113 1642 G G C noncoding n/d n/an/a n/a 47 7523965 1394758F6 SNP00100133 209 491 A A G M164 n/d n/d n/dn/d 47 7523965 1394758T6 SNP00033242 125 1605 G G C noncoding n/d n/an/a n/a 47 7523965 1631511T6 SNP00033242 186 1543 G G C noncoding n/dn/a n/a n/a 47 7523965 1964258H1 SNP00033242 56 1542 G G C noncoding n/dn/a n/a n/a 47 7523965 1964258H1 SNP00136906 186 1672 C C T noncodingn/a n/a n/a n/a 47 7523965 3149675H1 SNP00057801 173 785 G G A noncodingn/d n/a n/a n/a 47 7523965 3149675H1 SNP00096467 177 789 G G C noncodingn/a n/a n/a n/a 47 7523965 6595879J1 SNP00075756 251 798 G G A noncodingn/a n/a n/a n/a 47 7523965 759508T6 SNP00033242 199 1553 G G C noncodingn/d n/a n/a n/a 47 7523965 7636827H1 SNP00033242 227 1534 G G Cnoncoding n/d n/a n/a n/a 47 7523965 7636827H1 SNP00136906 97 1664 C C Tnoncoding n/a n/a n/a n/a 47 7523965 7637976J1 SNP00075756 569 543 G G Astop181 n/a n/a n/a n/a 51 7516229 1329019T6 SNP00069933 170 1162 G T Gnoncoding n/a n/a n/a n/a 51 7516229 6555450H1 SNP00023019 310 656 G G AS200 n/a n/a n/a n/a 52 7516525 2190612H1 SNP00128124 49 1276 A G A E413n/a n/a n/a n/a 52 7516525 3780651H1 SNP00074470 124 1682 C C Tnoncoding 0.95 0.96 0.77 0.65 52 7516525 3825922H1 SNP00074469 151 1667C C T S543 n/d n/d n/d n/d 53 7516533 000364H1 SNP00002194 28 1573 G A Gnoncoding 0.78 n/a n/a n/a 53 7516533 2360696T6 SNP00002194 447 1574 G AG noncoding 0.78 n/a n/a n/a 53 7516533 2641486F6 SNP00151695 95 1482 AA G noncoding n/a n/a n/a n/a 53 7516533 3078274H1 SNP00126890 34 1112 AA G P350 n/a n/a n/a n/a 53 7516533 3505057H1 SNP00126889 33 1081 A A GE340 n/a n/a n/a n/a 53 7516533 3505057H1 SNP00127060 80 1128 C C A R356n/a n/a n/a n/a 53 7516533 3505057H1 SNP00127061 247 1295 A A G E411 n/an/a n/a n/a 53 7516533 3505057H1 SNP00151694 133 1181 T T G P373 n/a n/an/a n/a 53 7516533 4376126H1 SNP00127062 27 1369 A A G K436 n/a n/a n/an/a 54 7516613 1741505T6 SNP00054334 116 2763 G G A R904 n/d n/d n/d n/d54 7516613 1741505T6 SNP00124224 52 2827 T T C S925 n/d n/d n/d n/d 547516613 1852144T6 SNP00029583 89 3808 C C T noncoding n/d n/a n/a n/a 547516613 2086173H1 SNP00029583 150 3791 C C T noncoding n/d n/a n/a n/a54 7516613 2103173R6 SNP00074035 137 331 A A G R93 n/d n/d n/d n/d 547516613 2172576F6 SNP00074035 62 327 A A G K92 n/d n/d n/d n/d 547516613 2230058H1 SNP00029582 57 2999 C C T H983 n/d n/a n/a n/a 547516613 2502887T6 SNP00029583 66 3831 C C T noncoding n/d n/a n/a n/a 547516613 2606210F6 SNP00029582 412 2998 C C T L982 n/d n/a n/a n/a 547516613 2606210F6 SNP00124225 348 2934 A A G K961 n/a n/a n/a n/a 547516613 2606210H1 SNP00054332 20 2607 G G A G852 n/a n/a n/a n/a 547516613 2606210H1 SNP00054333 135 2722 G G A R890 n/d n/a n/a n/a 547516613 2827761H1 SNP00124223 98 644 G G A E198 n/d n/d n/d n/d 547516613 3136587H1 SNP00124225 104 2935 A A G T961 n/a n/a n/a n/a 547516613 5971646H1 SNP00074036 28 1396 A G A E448 n/d n/d n/d n/d 547516613 5971646H1 SNP00074037 419 1786 C G C S578 n/a n/a n/a n/a 547516613 6203324H1 SNP00074038 314 1788 T T C L579 n/a n/a n/a n/a 547516613 7367225H1 SNP00098419 491 1864 A C A S604 0.86 n/d n/d n/d 557517068 201783T6 SNP00127935 369 3700 T T C noncoding n/a n/a n/a n/a 557517068 2836623F6 SNP00067424 105 2443 G G A G807 n/a n/a n/a n/a 557517068 2836623H1 SNP00067424 105 2442 G G A A807 n/a n/a n/a n/a 557517068 3003208F6 SNP00115029 381 640 A A G Q206 n/a n/a n/a n/a 557517068 6118733H1 SNP00115032 336 2129 C C T S702 n/d n/a n/a n/a 557517068 6448726H1 SNP00115031 288 1428 A A G I469 n/d n/a n/a n/a 557517068 6987676H1 SNP00115029 331 641 A A G P206 n/a n/a n/a n/a 557517068 7205376H1 SNP00115030 297 767 T T C N248 0.65 0.49 0.87 0.64 557517068 7649056H2 SNP00067424 283 2441 G G A A806 n/a n/a n/a n/a 567517148 1301060F6 SNP00028255 178 2035 G G C noncoding 0.99 n/d n/d n/d56 7517148 1436470H1 SNP00028255 92 2091 C G C noncoding 0.99 n/d n/dn/d 56 7517148 2008763H1 SNP00122615 72 2814 A A C noncoding n/a n/a n/an/a 56 7517148 2487070T6 SNP00122615 351 2839 A A C noncoding n/a n/an/a n/a 56 7517148 2504377T6 SNP00122615 334 2857 A A C noncoding n/an/a n/a n/a 56 7517148 2747152T6 SNP00067260 70 2411 A A C noncoding n/an/a n/a n/a 56 7517148 2836570F6 SNP00067260 348 2366 A A C noncodingn/a n/a n/a n/a 57 7517238 055029H1 SNP00035691 30 998 T T C S288 n/an/a n/a n/a 60 7520428 1006039H1 SNP00029126 28 4802 A A G noncoding n/dn/d n/d 0.99 60 7520428 1006039H1 SNP00134113 62 4836 G G C noncodingn/a n/a n/a n/a 60 7520428 1336820H1 SNP00100525 156 3711 C C Tnoncoding n/a n/a n/a n/a 60 7520428 1342990T6 SNP00029126 341 4818 A AG noncoding n/d n/d n/d 0.99 60 7520428 1349339F6 SNP00100526 22 3907 CC T noncoding n/a n/a n/a n/a 60 7520428 1349339F6 SNP00100527 61 3946 CC A noncoding n/a n/a n/a n/a 60 7520428 1501365F6 SNP00006017 178 2523A A G noncoding n/a n/a n/a n/a 60 7520428 1547712H1 SNP00029124 1053306 G G A noncoding n/a n/a n/a n/a 60 7520428 1547712H1 SNP00061149173 3374 A A G noncoding n/a n/a n/a n/a 60 7520428 1708824T6SNP00134113 303 4877 G G C noncoding n/a n/a n/a n/a 60 75204281812674H1 SNP00117686 180 4713 C C T noncoding 0.99 0.96 0.95 0.96 607520428 1861030T6 SNP00029126 368 4815 A A G noncoding n/d n/d n/d 0.9960 7520428 1861030T6 SNP00134113 334 4849 G G C noncoding n/a n/a n/an/a 60 7520428 1903968H1 SNP00029125 37 4436 A A G noncoding n/a n/a n/an/a 60 7520428 2345238T6 SNP00029126 377 4803 A A G noncoding n/d n/dn/d 0.99 60 7520428 2345238T6 SNP00117686 466 4714 C C T noncoding 0.990.96 0.95 0.96 60 7520428 2345238T6 SNP00134113 343 4837 G G C noncodingn/a n/a n/a n/a 60 7520428 2479846H1 SNP00136971 133 2478 T T Gnoncoding n/a n/a n/a n/a 60 7520428 264357T6 SNP00029126 362 4831 A A Gnoncoding n/d n/d n/d 0.99 60 7520428 264357T6 SNP00134113 328 4865 G GC noncoding n/a n/a n/a n/a 60 7520428 2792180T6 SNP00134113 339 4840 GG C noncoding n/a n/a n/a n/a 60 7520428 3699304H1 SNP00100524 54 3440 GG A noncoding n/a n/a n/a n/a 60 7520428 3790573H1 SNP00013785 201 4056T C T noncoding 0.23 n/a n/a n/a 60 7520428 4534017T1 SNP00134113 3244854 G G C noncoding n/a n/a n/a n/a 60 7520428 6388557H1 SNP00100523201 1774 C C T L579 n/a n/a n/a n/a 60 7520428 6831230J1 SNP00029126 2524827 A A G noncoding n/d n/d n/d 0.99 60 7520428 6831230J1 SNP00117686163 4738 C C T noncoding 0.99 0.96 0.95 0.96 60 7520428 6831230J1SNP00134113 286 4861 C G C noncoding n/a n/a n/a n/a 60 75204287688277H1 SNP00100525 219 3712 C C T noncoding n/a n/a n/a n/a 607520428 7689549J1 SNP00006017 344 2522 A A G noncoding n/a n/a n/a n/a60 7520428 7689549J1 SNP00136971 389 2477 T T G noncoding n/a n/a . n/an/a 60 7520428 7703916H1 SNP00029124 221 3307 G G A noncoding n/a n/an/a n/a 60 7520428 7703916H1 SNP00100524 82 3441 G G A noncoding n/a n/an/a n/a 60 7520428 7712761H1 SNP00029125 196 4437 A A G noncoding n/an/a n/a n/a 60 7520428 7756221J1 SNP00006017 74 2524 A A G noncoding n/an/a n/a n/a 60 7520428 7756221J1 SNP00136971 119 2479 T T G noncodingn/a n/a n/a n/a 60 7520428 990784R6 SNP00013785 260 4057 C C T noncoding0.23 n/a n/a n/a 61 7522586 1242156H1 SNP00114359 113 1870 G G Anoncoding n/a n/a n/a n/a 61 7522586 1285002H1 SNP00049573 23 1642 T T Gnoncoding 0.7 0.52 0.44 0.57 61 7522586 1342006H1 SNP00008735 103 375 CT C R57 n/a n/a n/a n/a 61 7522586 1377565F6 SNP00039374 123 2054 C C Tnoncoding n/d n/d n/d n/d 61 7522586 1377565F6 SNP00047780 66 1997 T T Cnoncoding n/a n/a n/a n/a 61 7522586 1377565F6 SNP00047781 77 2008 T C Tnoncoding n/a n/a n/a n/a 61 7522586 1377565H1 SNP00039374 123 2053 C CT noncoding n/d n/d n/d n/d 61 7522586 1377565H1 SNP00047780 66 1996 T TC noncoding n/a n/a n/a n/a 61 7522586 1377565H1 SNP00047781 77 2007 T CT noncoding n/a n/a n/a n/a 61 7522586 1377565T6 SNP00047780 510 2003 TT C noncoding n/a n/a n/a n/a 61 7522586 1377565T6 SNP00047781 499 2014T C T noncoding n/a n/a n/a n/a 61 7522586 1705173H1 SNP00018770 38 1406A A G noncoding n/d n/d n/d n/d 61 7522586 1857852H1 SNP00092603 141 631G A G noncoding 0.14 n/d 0.19 0.17 61 7522586 2108516H1 SNP00154337 44169 A A C noncoding n/a n/a n/a n/a 61 7522586 2655085H1 SNP00065632 191212 T T G G2 n/a n/a n/a n/a 61 7522586 2889783H1 SNP00152262 77 278 G GA K24 n/a n/a n/a n/a 61 7522586 3861045H1 SNP00059143 75 2014 A G Anoncoding 0.59 n/a n/a n/a 61 7522586 3948090T6 SNP00036245 44 1239 C TC noncoding n/a n/a n/a n/a 61 7522586 3948090T6 SNP00126649 97 1186 G GA noncoding n/a n/a n/a n/a 61 7522586 4550659T1 SNP00152262 306 258 G GA E18 n/a n/a n/a n/a 62 7524017 055029H1 SNP00035691 30 981 T T C S297n/a n/a n/a n/a 63 7525773 1274616F6 SNP00007308 253 167 G G A E50 0.87n/a n/a n/a 63 7525773 2608313H1 SNP00007308 169 172 G G A S52 0.87 n/an/a n/a 63 7525773 4435787F7 SNP00032647 519 847 G C G S277 n/d n/a n/an/a 65 2509577 5546336F7 SNP00070606 44 2484 T T C V770 n/a n/a n/a n/a67 7524408 2749757H1 SNP00121108 213 1411 T T C Y460 n/a n/a n/a n/a 677524408 4739562R7 SNP00033062 268 1957 C C T noncoding n/a n/a n/a n/a69 7526158 5206370H1 SNP00130724 195 667 G G C G211 n/a n/a n/a n/a 697526158 7960593H1 SNP00071326 132 3236 C T C G1067 0.46 0.37 0.67 0.4671 7526180 1458121H1 SNP00146630 133 2374 T T G noncoding n/a n/a n/an/a 71 7526180 1458121H1 SNP00146631 149 2390 G G A noncoding n/a n/an/a n/a 71 7526180 1663635F6 SNP00040633 39 1961 A A G noncoding 0.12n/a n/a n/a 71 7526180 1663635F6 SNP00040634 19 1981 A A C noncoding n/an/a n/a n/a 71 7526180 2013516T6 SNP00007120 336 419 C C T T1 0.67 n/an/a n/a 71 7526180 2013516T6 SNP00049608 105 188 C G C noncoding n/a n/an/a n/a 71 7526180 2254891H1 SNP00048399 135 961 C C T L182 n/a n/a n/an/a 71 7526180 2254891H1 SNP00096777 60 1036 G 0 T V207 0.88 n/a n/a n/a71 7526180 2254891R6 SNP00127250 274 822 G A G M135 n/a n/a n/a n/a 717526180 257026H1 SNP00007121 25 2206 A G A noncoding 0.15 n/a n/a n/a 717526180 257026H1 SNP00096771 58 2239 A A G noncoding n/a n/a n/a n/a 717526180 4712047F6 SNP00096075 2 1290 T T C S291 n/d n/a n/a n/a 717526180 6094776H1 SNP00146629 5 1875 A A G E486 n/a n/a n/a n/a 717526180 6355618F6 SNP00102652 224 231 C C T noncoding 0.42 0.44 0.450.46 71 7526180 6355618F6 SNP00148688 356 99 T C T noncoding n/a n/a n/an/a 71 7526180 6731321H1 SNP00096773 126 1600 A G A K395 0.74 0.91 0.310.76 72 7526185 125901F1 SNP00047602 255 3028 T C T noncoding 0.61 0.470.61 0.61 72 7526185 1553407H1 SNP00155225 129 1488 T T C noncoding n/an/a n/a n/a 72 7526185 2197671T6 SNP00155225 216 1533 T T C noncodingn/a n/a n/a n/a 72 7526185 6723530H1 SNP00051188 262 2713 A G Anoncoding n/a n/a n/a n/a 72 7526185 829638T6 SNP00155225 181 1582 C T Cnoncoding n/a n/a n/a n/a 73 7526192 1208904H1 SNP00062572 151 2429 G GA noncoding n/a n/a n/a n/a 73 7526192 1223444H1 SNP00098139 99 2262 C CT noncoding n/d n/a n/a n/d 73 7526192 1231274R6 SNP00115694 8 1018 C CT S113 n/a n/a n/a n/a 73 7526192 1341206H1 SNP00068492 33 1909 A A Gnoncoding n/d n/a n/a n/a 73 7526192 1405367T6 SNP00062572 57 2446 G G Anoncoding n/a n/a n/a n/a 73 7526192 1405367T6 SNP00098139 224 2279 C CT noncoding n/d n/a n/a n/d 73 7526192 1417137T6 SNP00062572 34 2482 G GA noncoding n/a n/a n/a n/a 73 7526192 1553058T6 SNP00062572 51 2453 G GA noncoding n/a n/a n/a n/a 73 7526192 1678219T6 SNP00098139 232 2271 CC T noncoding n/d n/a n/a n/d 73 7526192 1722718F6 SNP00068491 24 1686 CC T noncoding n/a n/a n/a n/a 73 7526192 1722718F6 SNP00068492 249 1911A A G noncoding n/d n/a n/a n/a 73 7526192 1722718H1 SNP00068491 24 1683C C T noncoding n/a n/a n/a n/a 73 7526192 2997552T6 SNP00062572 1802342 G G A noncoding n/a n/a n/a n/a 73 7526192 2997552T6 SNP00098139347 2177 C C T noncoding n/d n/a n/a n/d 73 7526192 7674218H2SNP00068492 342 1908 A A G noncoding n/d n/a n/a n/a 74 75261931328791H1 SNP00057788 217 716 G G T noncoding n/a n/a n/a n/a 74 75261934291033F6 SNP00142508 164 1539 A A G N232 n/a n/a n/a n/a 74 75261934291033F6 SNP00142509 191 1566 A A G T241 n/a n/a n/a n/a 74 75261937217965H1 SNP00118120 200 145 G A G noncoding n/d n/a n/a n/a 74 75261937760201H1 SNP00057788 118 715 G G T noncoding n/a n/a n/a n/a 75 75261961216956H1 SNP00006796 60 4315 A A G noncoding 0.98 n/a n/a n/a 757526196 1224406H1 SNP00124328 162 2487 A A G noncoding n/d n/a n/a n/a75 7526196 1436210H1 SNP00006288 103 3533 G G A noncoding 0.97 n/a n/an/a 75 7526196 1438205H1 SNP00124330 102 3121 A A G noncoding n/d n/an/a n/a 75 7526196 1555235H1 SNP00006287 133 3401 C T C noncoding 0.34n/a n/a n/a 75 7526196 1597263F6 SNP00124329 113 2824 A A G noncodingn/d n/d n/d n/d 75 7526196 1669032H1 SNP00124327 46 65 C T C noncodingn/a n/a n/a n/a 75 7526196 2555446F6 SNP00068980 190 1856 A A Gnoncoding n/d n/d n/d n/d 75 7526196 2555446H1 SNP00068980 190 1854 A AG noncoding n/d n/d n/d n/d 75 7526196 3337906H1 SNP00153438 134 2187 GG A noncoding n/a n/a n/a n/a 75 7526196 3643184H1 SNP00068979 155 1594C C T noncoding n/a n/a n/a n/a 75 7526196 6754284H1 SNP00068978 4201195 A A G noncoding n/a n/a n/a n/a 75 7526196 7703764J1 SNP00124330162 3128 A A G noncoding n/d n/a n/a n/a 75 7526196 7753868H1SNP00124329 510 2852 A A G noncoding n/d n/d n/d n/d 75 75261967753868H1 SNP00124330 213 3149 A A G noncoding n/d n/a n/a n/a 757526196 8598525H1 SNP00006287 238 3428 T T C noncoding 0.34 n/a n/a n/a75 7526196 8598525H1 SNP00006288 370 3560 G G A noncoding 0.97 n/a n/an/a 76 7526198 1216956H1 SNP00006796 60 4122 A A G K1309 0.98 n/a n/an/a 76 7526198 1224406H1 SNP00124328 162 2284 A A G K696 n/d n/a n/a n/a76 7526198 1436210H1 SNP00006288 103 3331 G G A A1045 0.97 n/a n/a n/a76 7526198 1438205H1 SNP00124330 102 2918 A A G T908 n/d n/a n/a n/a 767526198 1555235H1 SNP00006287 133 3199 C T C S1001 0.34 n/a n/a n/a 767526198 1597263F6 SNP00124329 113 2621 A A G N809 n/d n/d n/d n/d 767526198 1669032H1 SNP00124327 46 65 C T C noncoding n/a n/a n/a n/a 767526198 1806969T6 SNP00029581 329 4272 C C T noncoding n/a n/a n/a n/a76 7526198 1922794H1 SNP00092542 84 4217 G G A D1341 n/a n/a n/a n/a 767526198 2005750H1 SNP00029581 61 4270 C C T noncoding n/a n/a n/a n/a 767526198 2189973H1 SNP00136926 25 4147 C C T A1317 n/a n/a n/a n/a 767526198 2555446F6 SNP00068980 190 1655 A A G N487 n/d n/d n/d n/d 767526198 2555446H1 SNP00068980 190 1653 A A G Y486 n/d n/d n/d n/d 767526198 2936740H1 SNP00006289 104 3928 C C T T1244 0.91 n/a n/a n/a 767526198 3337906H1 SNP00153438 134 1984 G G A R596 n/a n/a n/a n/a 767526198 3643184H1 SNP00068979 155 1393 C C T S399 n/a n/a n/a n/a 767526198 6754284H1 SNP00068978 420 994 A A G G266 n/a n/a n/a n/a 767526198 7703764J1 SNP00124330 162 2925 A A G Q910 n/d n/a n/a n/a 767526198 7753868H1 SNP00124329 510 2649 A A G D818 n/d n/d n/d n/d 767526198 7753868H1 SNP00124330 213 2946 A A G H917 n/d n/a n/a n/a 767526198 8598525H1 SNP00006287 238 3226 T T C D1010 0.34 n/a n/a n/a 767526198 8598525H1 SNP00006288 370 3358 G G A T1054 0.97 n/a n/a n/a 777526208 1236920F1 SNP00033469 330 3805 C C T noncoding n/a n/a n/a n/a77 7526208 1284901H1 SNP00013862 33 2502 C C G Q454 0.91 n/a n/a n/a 777526208 1915448H1 SNP00003491 119 5753 A A G noncoding n/a n/a n/a n/a77 7526208 2528372H1 SNP00053975 148 2683 C C T noncoding n/a n/a n/an/a 77 7526208 2681418H1 SNP00053974 116 2185 T T C L348 n/d n/a n/a n/a77 7526208 2749684F6 SNP00153340 187 1794 T T C W218 n/a n/a n/a n/a 777526208 3331418H1 SNP00132658 181 2866 T T C noncoding n/a n/a n/a n/a77 7526208 3693823H1 SNP00053972 67 1115 C C T noncoding n/d n/a n/a n/a77 7526208 3967421F6 SNP00033469 163 3806 C C T noncoding n/a n/a n/an/a 77 7526208 5055874H1 SNP00113323 139 5405 T T G noncoding n/a n/an/a n/a 77 7526208 6449431H1 SNP00053973 480 1969 T C T V276 n/a n/a n/an/a 78 7526212 2749684F6 SNP00153340 187 1794 T T C W218 n/a n/a n/a n/a78 7526212 3693823H1 SNP00053972 67 1115 C C T noncoding n/d n/a n/a n/a78 7526212 6449431H1 SNP00053973 480 1969 T C T V276 n/a n/a n/a n/a 797526213 1430148F6 SNP00014900 123 3197 C G C noncoding 0.04 n/a n/a n/a79 7526213 2113230H1 SNP00044591 151 6811 C C T noncoding n/a n/a n/an/a 79 7526213 2113230R6 SNP00044591 151 6814 C C T noncoding n/a n/an/a n/a 79 7526213 2556574H1 SNP00139931 137 2468 A A G noncoding n/an/a n/a n/a 79 7526213 2987033F6 SNP00153180 352 6398 C C T noncodingn/a n/a n/a n/a 79 7526213 3844660H1 SNP00014900 64 3198 C G C noncoding0.04 n/a n/a n/a 79 7526213 712904R6 SNP00139930 72 2210 A G A noncodingn/a n/a n/a n/a 80 7526214 1430148F6 SNP00014900 123 3134 C G Cnoncoding 0.04 n/a n/a n/a 80 7526214 2113230H1 SNP00044591 151 6746 C CT noncoding n/a n/a n/a n/a 80 7526214 2113230R6 SNP00044591 151 6749 CC T noncoding n/a n/a n/a n/a 80 7526214 2556574H1 SNP00139931 137 2403A A G noncoding n/a n/a n/a n/a 80 7526214 2987033F6 SNP00153180 3526333 C C T noncoding n/a n/a n/a n/a 80 7526214 3844660H1 SNP00014900 643135 C G C noncoding 0.04 n/a n/a n/a 81 7526228 1485690T6 SNP00066816319 2716 G G A noncoding n/d n/d 0.96 n/d 81 7526228 1835249H1SNP00066816 8 2712 G G A noncoding n/d n/d 0.96 n/d 81 7526228 2169542T6SNP00066816 241 2740 G G A noncoding n/d n/d 0.96 n/d 81 75262282536771H1 SNP00136441 18 234 G G A noncoding n/a n/a n/a n/a 81 75262282805663T6 SNP00066816 251 2787 G G A noncoding n/d n/d 0.96 n/d 817526228 510019T6 SNP00066816 194 2713 G G A noncoding n/d n/d 0.96 n/d82 7526246 1294154H1 SNP00068998 123 2362 A A G noncoding n/a n/a n/an/a 82 7526246 1545488H1 SNP00068997 78 2115 C C G noncoding n/a n/a n/an/a 82 7526246 280325T6 SNP00068997 58 2182 C C G noncoding n/a n/a n/an/a 82 7526246 4407121H1 SNP00041996 224 2946 A A G noncoding n/a n/an/a n/a 82 7526246 7621966J1 SNP00068997 165 2116 C C G noncoding n/an/a n/a n/a 82 7526246 7751044H1 SNP00068998 486 2365 A A G noncodingn/a n/a n/a n/a 83 7526258 1348638F6 SNP00076027 241 328 G G C G74 n/dn/d n/d n/d 83 7526258 1348638F6 SNP00132757 63 150 A A G R14 n/a n/an/a n/a 83 7526258 1444773H1 SNP00037439 67 402 G G C L98 n/a n/a n/an/a 83 7526258 1897166H1 SNP00043983 191 1194 T T C P362 n/a n/a n/a n/a83 7526258 2770947H1 SNP00154171 21 1113 C T C D335 n/a n/a n/a n/a 837526258 3143852H1 SNP00037440 73 1151 T C T L348 n/d n/d n/d n/d 837526258 3143852H1 SNP00111294 30 1108 C C T L334 1 n/d n/d n/d 847526311 1649261F6 SNP00019740 300 2179 T T C noncoding n/a n/a n/a n/a84 7526311 1649261T6 SNP00019740 252 2259 T T C noncoding n/a n/a n/an/a 84 7526311 268900T6 SNP00019740 257 2257 T T C noncoding n/a n/a n/an/a 84 7526311 2745158H1 SNP00058093 32 1282 T T C noncoding 0.82 0.90.98 0.92 84 7526311 2745158H1 SNP00114001 97 1347 G T G noncoding n/an/a n/a n/a 84 7526311 2921293T6 SNP00019740 268 2235 T T C noncodingn/a n/a n/a n/a 84 7526311 8011285H1 SNP00125603 438 540 C C T A129 n/an/a n/a n/a 85 7526315 058064H1 SNP00003740 186 1775 A A G noncoding n/an/a n/a n/a 85 7526315 1004004H1 SNP00012539 79 5335 T C T noncoding n/an/a n/a n/a 85 7526315 1004004H1 SNP00012540 191 5447 G A G noncoding0.71 0.63 0.86 0.64 85 7526315 1004004H1 SNP00045700 205 5461 C C Tnoncoding n/a n/a n/a n/a 85 7526315 1330039H1 SNP00045701 48 5601 G G Cnoncoding n/a n/a n/a n/a 85 7526315 1363254H1 SNP00022215 65 2848 A A Cnoncoding n/d n/a n/a n/a 85 7526315 1377277F1 SNP00012538 87 4783 C C Tnoncoding n/a n/a n/a n/a 85 7526315 1675313F6 SNP00028237 82 3316 T T Cnoncoding n/a n/a n/a n/a 85 7526315 1675313F6 SNP00028238 133 3367 A GA noncoding n/d n/a n/a n/a 85 7526315 1675313T6 SNP00012538 16 4780 C CT noncoding n/a n/a n/a n/a 85 7526315 1682961T7 SNP00045701 458 5606 GG C noncoding n/a n/a n/a n/a 85 7526315 3003741H1 SNP00023889 140 215 TT G G5 n/a n/a n/a n/a 85 7526315 403838T6 SNP00028238 336 3387 G G Anoncoding n/d n/a n/a n/a 85 7526315 7622837J1 SNP00028237 242 3317 T TC noncoding n/a n/a n/a n/a 85 7526315 7622837J1 SNP00028238 191 3368 GG A noncoding n/d n/a n/a n/a 85 7526315 7625836H1 SNP00028237 72 3299 TT C noncoding n/a n/a n/a n/a 85 7526315 7625836H1 SNP00028238 123 3348G G A noncoding n/d n/a n/a n/a 85 7526315 7752327H1 SNP00045701 5175600 G G C noncoding n/a n/a n/a n/a 86 7526442 1265917F1 SNP00149600355 1648 T T C noncoding n/a n/a n/a n/a 86 7526442 1382145F6SNP00149600 469 1646 T T C noncoding n/a n/a n/a n/a 86 75264421824201F6 SNP00066979 79 724 C C T S139 n/d n/d n/a n/d 86 75264422046231H1 SNP00114113 226 1004 G G C noncoding n/d n/a n/a n/a 867526442 2744627F6 SNP00022802 98 167 C C G noncoding n/a n/a n/a n/a 867526442 691185T6 SNP00149600 55 1722 T T C noncoding n/a n/a n/a n/a 867526442 7622751H1 SNP00031991 177 769 C C T noncoding n/a n/a n/a n/a

1. An isolated polynucleotide encoding a polypeptide, wherein thepolypeptide consists of the amino acid sequence of SEQ ID NO:
 13. 2. AnThe isolated polynucleotide of claim 1 wherein the polynucleotidesequence consists of SEQ ID NO:
 56. 3. A recombinant polynucleotidecomprising a promoter sequence operably linked to the polynucleotide ofclaim
 1. 4. An isolated cell transformed with the recombinantpolynucleotide of claim
 3. 5. An isolated polynucleotide complementaryto the polynucleotide of claim
 1. 6. An isolated polynucleotidecomplementary to the polynucleotide of claim
 2. 7. An RNA equivalent ofthe polynucleotide of claim 1.